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Structural and biochemical characterization of a metagenome-derived esterase with a long N-terminal extension.
Protein Sci.
PUBLISHED: 09-25-2014
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The genes encoding six novel esterolytic/lipolytic enzymes, termed LC-Est1˜6, were isolated from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. These enzymes greatly vary in size and amino acid sequence. The highest identity between the amino acid sequence of each enzyme and that available from the database varies from 44 to 73%. Of these metagenome-derived enzymes, LC-Est1 is characterized by the presence of a long N-terminal extension (LNTE, residues 26-283) between a putative signal peptide (residues 1-25) and a C-terminal esterase domain (residues 284-510). A putative esterase from Candidatus Solibacter usitatus (CSu-Est) is the only protein which shows the significant amino acid sequence identity (46%) to the entire region of LC-Est1. To examine whether LC-Est1 exhibits activity and its LNTE is important for activity and stability of the esterase domain, LC-Est1 (residues 26-510), LC-Est1C (residues 284-510) and LC-Est1C* (residues 304-510) were overproduced in E. coli, purified and characterized. LC-Est1C* was only used for structural analysis. The crystal structure of LC-Est1C* highly resembles that of the catalytic domain of Thermotoga maritima esterase (Tm-EstA), suggesting that LNTE is not required for folding of the esterase domain. The enzymatic activity of LC-Est1C was lower than that of LC-Est1 by 60%, although its substrate specificity was similar to that of LC-Est1. LC-Est1C was less stable than LC-Est1 by 3.3°C. These results suggest that LNTE of LC-Est1 rather exists as an independent domain but is required for maximal activity and stability of the esterase domain.
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Rational design of a glycosynthase by the crystal structure of ?-galactosidase from Bacillus circulans (BgaC) and its use for the synthesis of N-acetyllactosamine type 1 glycan structures.
J. Biotechnol.
PUBLISHED: 03-27-2014
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The crystal structure of ?-galactosidase from Bacillus circulans (BgaC) was determined at 1.8? resolution. The overall structure of BgaC consists of three distinct domains, which are the catalytic domain with a TIM-barrel structure and two all-? domains (ABDs). The main-chain fold and steric configurations of the acidic and aromatic residues at the active site were very similar to those of Streptococcus pneumoniae ?(1,3)-galactosidase BgaC in complex with galactose. The structure of BgaC was used for the rational design of a glycosynthase. BgaC belongs to the glycoside hydrolase family 35. The essential nucleophilic amino acid residue has been identified as glutamic acid at position 233 by site-directed mutagenesis. Construction of the active site mutant BgaC-Glu233Gly gave rise to a galactosynthase transferring the sugar moiety from ?-d-galactopyranosyl fluoride (?GalF) to different ?-linked N-acetylglucosamine acceptor substrates in good yield (40-90%) with a remarkably stable product formation. Enzymatic syntheses with BgaC-Glu233Gly afforded the stereo- and regioselective synthesis of ?1-3-linked key galactosides like galacto-N-biose or lacto-N-biose.
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Crystal structure and thermodynamic and kinetic stability of metagenome-derived LC-cutinase.
Biochemistry
PUBLISHED: 03-13-2014
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The crystal structure of metagenome-derived LC-cutinase with polyethylene terephthalate (PET)-degrading activity was determined at 1.5 Å resolution. The structure strongly resembles that of Thermobifida alba cutinase. Ser165, Asp210, and His242 form the catalytic triad. Thermal denaturation and guanidine hydrochloride (GdnHCl)-induced unfolding of LC-cutinase were analyzed at pH 8.0 by circular dichroism spectroscopy. The midpoint of the transition of the thermal denaturation curve, T1/2, and that of the GdnHCl-induced unfolding curve, Cm, at 30 °C were 86.2 °C and 4.02 M, respectively. The free energy change of unfolding in the absence of GdnHCl, ?G(H2O), was 41.8 kJ mol(-1) at 30 °C. LC-cutinase unfolded very slowly in GdnHCl with an unfolding rate, ku(H2O), of 3.28 × 10(-6) s(-1) at 50 °C. These results indicate that LC-cutinase is a kinetically robust protein. Nevertheless, the optimal temperature for the activity of LC-cutinase toward p-nitrophenyl butyrate (50 °C) was considerably lower than the T1/2 value. It increased by 10 °C in the presence of 1% polyethylene glycol (PEG) 1000. It also increased by at least 20 °C when PET was used as a substrate. These results suggest that the active site is protected from a heat-induced local conformational change by binding of PEG or PET. LC-cutinase contains one disulfide bond between Cys275 and Cys292. To examine whether this disulfide bond contributes to the thermodynamic and kinetic stability of LC-cutinase, C275/292A-cutinase without this disulfide bond was constructed. Thermal denaturation studies and equilibrium and kinetic studies of the GdnHCl-induced unfolding of C275/292A-cutinase indicate that this disulfide bond contributes not only to the thermodynamic stability but also to the kinetic stability of LC-cutinase.
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Structural basis for salt-dependent folding of ribonuclease H1 from halophilic archaeon Halobacterium sp. NRC-1.
J. Struct. Biol.
PUBLISHED: 02-26-2014
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RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1 (Halo-RNase H1) requires ?2M NaCl, ?10mM MnCl2, or ?300mM MgCl2 for folding. To understand the structural basis for this salt-dependent folding of Halo-RNase H1, the crystal structure of Halo-RNase H1 was determined in the presence of 10mM MnCl2. The structure of Halo-RNase H1 highly resembles those of metagenome-derived LC11-RNase H1 and Sulfolobus tokodaii RNase H1 (Sto-RNase H1), except that it contains two Mn(2+) ions at the active site and has three bi-aspartate sites on its surface. To examine whether negative charge repulsion at these sites are responsible for low-salt denaturation of Halo-RNase H1, a series of the mutant proteins of Halo-RNase H1 at these sites were constructed. The far-UV CD spectra of these mutant proteins measured in the presence of various concentrations of NaCl suggest that these mutant proteins exist in an equilibrium between a partially folded state and a folded state. However, the fraction of the protein in a folded state is nearly 0% for the active site mutant, 40% for the bi-aspartate site mutant, and 70% for the mutant at both sites in the absence of salt. The active site mutant requires relatively low concentration (?0.5M) of salt for folding. These results suggest that suppression of negative charge repulsion at both active and bi-aspartate sites by salt is necessary to yield a folded protein.
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Structural and mechanistic insights into the kynurenine aminotransferase-mediated excretion of kynurenic acid.
J. Struct. Biol.
PUBLISHED: 01-18-2014
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Kynurenine aminotransferase (KAT) is a homodimeric pyridoxal protein that mediates the catalytic conversion of kynurenine (KYN) to kynurenic acid (KYA), an endogenous N-methyl-d-aspartate (NMDA) receptor antagonist. KAT is involved in the biosynthesis of glutamic and aspartic acid, functions as a neurotransmitter for the NMDA receptor in mammals, and is regulated by allosteric mechanisms. Its importance in various diseases such as schizophrenia makes KAT a highly attractive drug target. Here, we present the crystal structure of the Pyrococcus horikoshii KAT (PhKAT) in complex with pyridoxamine phosphates (PMP), KYN, and KYA. Surprisingly, the PMP was bound to the LYS-269 of phKAT by forming a covalent hydrazine bond. This crystal structure clearly shows that an amino group of KYN was transaminated to PLP, which forms a Schiff's base with the LYS-269 of the KYN. Thus, our structure confirms that the PMPs represent an intermediate state during the KAT reaction. Thus, PhKAT catalyzes the sequential conversion of KYN to KYA via the formation of an intermediate 4-(2-aminophenyl)-2,4-dioxobutanoate (4AD), which is spontaneously converted to KYA in the absence of an amino group acceptor. Furthermore, we identified the two entry and exit sites of the PhKAT homodimer for KYN and KYA, respectively. The structural data on PhKAT presented in this manuscript contributes to further the understanding of transaminase enzyme reaction mechanisms.
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Divalent metal ion-induced folding mechanism of RNase H1 from extreme halophilic archaeon Halobacterium sp. NRC-1.
PLoS ONE
PUBLISHED: 01-01-2014
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RNase H1 from Halobacterium sp. NRC-1 (Halo-RNase H1) is characterized by the abundance of acidic residues on the surface, including bi/quad-aspartate site residues. Halo-RNase H1 exists in partially folded (I) and native (N) states in low-salt and high-salt conditions respectively. Its folding is also induced by divalent metal ions. To understand this unique folding mechanism of Halo-RNase H1, the active site mutant (2A-RNase H1), the bi/quad-aspartate site mutant (6A-RNase H1), and the mutant at both sites (8A-RNase H1) were constructed. The far-UV CD spectra of these mutants suggest that 2A-RNase H1 mainly exists in the I state, 6A-RNase H1 exists both in the I and N states, and 8A-RNase H1 mainly exists in the N state in a low salt-condition. These results suggest that folding of Halo-RNase H1 is induced by binding of divalent metal ions to the bi/quad-aspartate site. To examine whether metal-induced folding is unique to Halo-RNase H1, RNase H2 from the same organism (Halo-RNase H2) was overproduced and purified. Halo-RNase H2 exists in the I and N states in low-salt and high-salt conditions respectively, as does Halo-RNase H1. However, this protein exists in the I state even in the presence of divalent metal ions. Halo-RNase H2 exhibits junction ribonuclease activity only in a high-salt condition. A tertiary model of this protein suggests that this protein does not have a quad-aspartate site. We propose that folding of Halo-RNase H1 is induced by binding of divalent metal ion to the quad-aspartate site in a low-salt condition.
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Formation of the High-Affinity Calcium Binding Site in Pro-subtilisin E with the Insertion Sequence IS1 of Pro-Tk-subtilisin.
Biochemistry
PUBLISHED: 12-04-2013
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Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability. This site is not formed in Pro-subtilisin E, because the structural rearrangement of the N-terminal region of the subtilisin domain upon autoprocessing is necessary for the formation of this site. As a result, Pro-subtilisin E is not fully folded. In contrast, Pro-Tk-subtilisin from Thermococcus kodakarensis is fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains. To examine whether the Ca1 site is formed in Pro-subtilisin E by inserting IS1 between the propeptide and subtilisin domains, the Pro-subtilisin E mutant with this insertion, IS1-Pro-subtilisin E, and its active site mutants, IS1-Pro-S221A and IS1-Pro-S221C, were constructed and characterized. The crystal structure of IS1-Pro-S221A revealed that this protein is fully folded and the Ca1 site is formed. In this structure, IS1 serves as a linker that brings the N-terminus of the subtilisin domain near the Ca1 site. IS1-Pro-S221A in a calcium-bound form was more stable than that in a calcium-free form by 13.1 °C. IS1-Pro-S221C was more rapidly autoprocessed than Pro-S221C. These results suggest that IS1 facilitates the formation of the Ca1 site and the complete folding of Pro-subtilisin E and thereby accelerates its autoprocessing.
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Increase in activation rate of Pro-Tk-subtilisin by a single nonpolar-to-polar amino acid substitution at the hydrophobic core of the propeptide domain.
Protein Sci.
PUBLISHED: 07-12-2013
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Tk-subtilisin (Gly70-Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk-subtilisin is produced from its inactive precursor, Pro-Tk-subtilisin (Gly1-Gly398), by autoprocessing and degradation of the propeptide (Tk-propeptide, Gly1-Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk-propeptide. Tk-propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar-to-polar amino acid substitution at this core affects the activation rate of Pro-Tk-subtilisin, the Pro-Tk-subtilisin derivative with the Phe17 ? His mutation (Pro-F17H), Tk-propeptide derivative with the same mutation (F17H-propeptide), and two active-site mutants of Pro-F17H (Pro-F17H/S324A and Pro-F17H/S324C) were constructed. The crystal structure of Pro-F17H/S324A was nearly identical to that of Pro-S324A, indicating that the mutation does not affect the structure of Pro-Tk-subtilisin. The refolding rate of Pro-F17H/S324A and autoprocessing rate of Pro-F17H/S324C were also nearly identical to those of their parent proteins (Pro-S324A and Pro-S324C). However, the activation rate of Pro-F17H greatly increased when compared with that of Pro-Tk-subtilisin, such that Pro-F17H is efficiently activated even at 40°C. The far-UV circular dichroism spectrum of F17H-propeptide did not exhibit a broad trough at 205-230 nm, which is observed in the spectrum of Tk-propeptide. F17H-propeptide is more susceptible to chymotryptic degradation than Tk-propeptide. These results suggest that F17H-propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk-subtilisin. Thus, destabilization of the hydrophobic core of Tk-propeptide by a nonpolar-to-polar amino acid substitution is an effective way to increase the activation rate of Pro-Tk-subtilisin.
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Evolvability of thermophilic proteins from archaea and bacteria.
Biochemistry
PUBLISHED: 07-03-2013
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Proteins from thermophiles possess high thermostability. The stabilization mechanisms differ between archaeal and bacterial proteins, whereby archaeal proteins are mainly stabilized via hydrophobic interactions and bacterial proteins by ion pairs. High stability is an important factor in promoting protein evolution, but the precise means by which different stabilization mechanisms affect the evolution process remain unclear. In this study, we investigated a random mutational drift of esterases from thermophilic archaea and bacteria at high temperatures. Our results indicate that mutations in archaeal proteins lead to improved function with no loss of stability, while mutant bacterial proteins are largely destabilized with decreased activity at high temperatures. On the basis of these findings, we suggest that archaeal proteins possess higher "evolvability" than bacterial proteins under temperature selection and are additionally able to evolve into eukaryotic proteins.
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Role of N-terminal extension of Bacillus stearothermophilus RNase H2 and C-terminal extension of Thermotoga maritima RNase H2.
FEBS J.
PUBLISHED: 06-02-2013
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Bacillus stearothermophilus RNase H2 (BstRNH2) and Thermotoga maritima RNase H2 (TmaRNH2) have N-terminal and C-terminal extensions, respectively, as compared with Aquifex aeolicus RNase H2 (AaeRNH2). To analyze the role of these extensions, BstRNH2 and TmaRNH2 without these extensions were constructed, and their biochemical properties were compared with those of their intact partners and AaeRNH2. The far-UV CD spectra of all proteins were similar, suggesting that the protein structure is not significantly altered by removal of these extensions. However, both the junction ribonuclease and RNase H activities of BstRNH2 and TmaRNH2, as well as their substrate-binding affinities, were considerably decreased by removal of these extensions. The stability of BstRNH2 and TmaRNH2 was also decreased by removal of these extensions. The activity, substrate binding affinity and stability of TmaRNH2 without the C-terminal 46 residues were partly restored by the attachment of the N-terminal extension of BstRNH2. These results suggest that the N-terminal extension of BstRNH2 functions as a substrate-binding domain and stabilizes the RNase H domain. Because the C-terminal extension of TmaRNH2 assumes a helix hairpin structure and does not make direct contact with the substrate, this extension is probably required to make the conformation of the substrate-binding site functional. AaeRNH2 showed comparable junction ribonuclease activity to those of BstRNH2 and TmaRNH2, and was more stable than these proteins, indicating that bacterial RNases H2 do not always require an N-terminal or C-terminal extension to increase activity, substrate-binding affinity, and/or stability.
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Investigating the structural dependence of protein stabilization by amino acid substitution.
Biochemistry
PUBLISHED: 04-12-2013
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A goal of protein engineering technology is developing methods to increase protein stability. However, rational design of stable proteins is difficult because the stabilization mechanism of proteins is not fully understood. In this study, we examined the structural dependence of protein stabilization by introducing single amino acid substitution into ribonuclease H1 from the psychotropic bacterium Shewanella oneidensis MR-1 (So-RNase H1), which was our model protein. We performed saturation mutagenesis at various sites. Mutations that stabilized So-RNase H1 were screened using an RNase H-dependent temperature-sensitive Escherchia coli strain. Stabilizing mutations were identified by the suppressor mutagenesis method. This method yielded 39 stabilized mutants from 513 mutations at 27 positions. This suggested that more than 90% of mutations caused destabilization even in a psychotropic protein. However, 17 positions had stabilizing mutations, indicating that the stabilization factors were dispersed over many positions. Interestingly, the identified mutations were distributed mainly at exposed or nonconserved sites. These results provide a novel strategy for protein stabilization.
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Proteolysis of abnormal prion protein with a thermostable protease from Thermococcus kodakarensis KOD1.
Appl. Microbiol. Biotechnol.
PUBLISHED: 03-14-2013
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The abnormal prion protein (scrapie-associated prion protein, PrP(Sc)) is considered to be included in the group of infectious agents of transmissible spongiform encephalopathies. Since PrP(Sc) is highly resistant to normal sterilization procedures, the decontamination of PrP(Sc) is a significant public health issue. In the present study, a hyperthermostable protease, Tk-subtilisin, was used to degrade PrP(Sc). Although PrP(Sc) is known to be resistant toward proteolytic enzymes, Tk-subtilisin was able to degrade PrP(Sc) under extreme conditions. The level of PrP(Sc) in brain homogenates was found to decrease significantly in vitro following Tk-subtilisin treatment at 100 °C, whereas some protease-resistant fractions remain after proteinase K treatment. Rather small amounts of Tk-subtilisin (0.3 U) were required to degrade PrP(Sc) at 100 °C and pH 8.0. In addition, Tk-subtilisin was observed to degrade PrP(Sc) in the presence of sodium dodecyl sulfate or other industrial surfactants. Although several proteases degrading PrP(Sc) have been reported, practical decontamination procedures using enzymes are not available. This report aims to provide basic information for the practical use of a proteolytic enzyme for PrP(Sc) degradation.
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Crystal structure of metagenome-derived LC11-RNase H1 in complex with RNA/DNA hybrid.
J. Struct. Biol.
PUBLISHED: 02-26-2013
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LC11-RNase H1 is a Sulfolobus tokodaii RNase H1 (Sto-RNase H1) homologue isolated by metagenomic approach. In this study, the crystal structure of LC11-RNase H1 in complex with an RNA/DNA substrate was determined. Unlike Bacillus halodurans RNase H1 without hybrid binding domain (HBD) (Bh-RNase HC) and human RNase H1 without HBD (Hs-RNase HC), LC11-RNase H1 interacts with four non-consecutive 2-OH groups of the RNA strand. The lack of interactions with four consecutive 2-OH groups leads to a dramatic decrease in the ability of LC11-RNase H1 to cleave the DNA-RNA-DNA/DNA substrate containing four ribonucleotides as compared to those to cleave the substrates containing five and six ribonucleotides. The interaction of LC11-RNase H1 with the DNA strand is also different from those of Bh-RNase HC and Hs-RNase HC. Beside the common phosphate-binding pocket, LC11-RNase H1 has a unique DNA-binding channel. Furthermore, the active-site residues of LC11-RNase H1 are located farther away from the scissile phosphate group than those of Bh-RNase HC and Hs-RNase HC. Modeling of Sto-RNase H1 in complex with the 14bp RNA/DNA substrate, together with the structure-based mutational analyses, suggest that the ability of Sto-RNase H1 to cleave double-stranded RNA is dependent on the local conformation of the basic residues located at the DNA binding site.
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Enzymatic activity of a subtilisin homolog, Tk-SP, from Thermococcus kodakarensis in detergents and its ability to degrade the abnormal prion protein.
BMC Biotechnol.
PUBLISHED: 02-26-2013
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Tk-SP is a member of subtilisin-like serine proteases from a hyperthermophilic archaeon Thermococcus kodakarensis. It has been known that the hyper-stable protease, Tk-SP, could exhibit enzymatic activity even at high temperature and in the presence of chemical denaturants. In this work, the enzymatic activity of Tk-SP was measured in the presence of detergents and EDTA. In addition, we focused to demonstrate that Tk-SP could degrade the abnormal prion protein (PrPSc), a protease-resistant isoform of normal prion protein (PrPC).
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Crystallization and X-ray structure determination of a thermoalkalophilic lipase from Geobacillus SBS-4S.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 02-20-2013
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A thermoalkalophilic lipase (LIPSBS) from the newly isolated Geobacillus strain SBS-4S which hydrolyzes a wide range of fatty acids has been characterized. In the present study, the crystallization of purified LIPSBS using the sitting-drop vapour-diffusion method and its X-ray diffraction studies are described. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 55.13, b = 71.75, c = 126.26 Å. The structure was determined at 1.6 Å resolution by the molecular-replacement method using the lipase from G. stearothermophilus L1 as a model.
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Crystal structure of metagenome-derived LC9-RNase H1 with atypical DEDN active site motif.
FEBS Lett.
PUBLISHED: 02-13-2013
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The crystal structure of metagenome-derived LC9-RNase H1 was determined. The structure-based mutational analyses indicated that the active site motif of LC9-RNase H1 is altered from DEDD to DEDN. In this motif, the location of the second glutamate residue is moved from ?A-helix to ?1-strand immediately next to the first aspartate residue, as in the active site of RNase H2. However, the structure and enzymatic properties of LC9-RNase H1 highly resemble those of RNase H1, instead of RNase H2. We propose that LC9-RNase H1 represents bacterial RNases H1 with an atypical DEDN active site motif, which are evolutionarily distinct from those with a typical DEDD active site motif.
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Accelerated maturation of Tk-subtilisin by a Leu?Pro mutation at the C-terminus of the propeptide, which reduces the binding of the propeptide to Tk-subtilisin.
FEBS J.
PUBLISHED: 01-11-2013
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Tk-subtilisin, a subtilisin homologue (Gly70-Gly398) from Thermococcus kodakarensis, is matured from its precursor, Pro-Tk-subtilisin [Tk-subtilisin in a pro form (Gly1-Gly398)], by autoprocessing and degradation of propeptide [Tk-propeptide, a propeptide of Tk-subtilisin (Gly1-Leu69)]. The scissile peptide bond between Leu69 and Gly70 of Pro-Tk-subtilisin is first self-cleaved to produce an inactive Tk-propeptide:Tk-subtilisin complex, in which the C-terminal region of Tk-propeptide binds to the active-site cleft of Tk-subtilisin. Tk-propeptide is then dissociated from Tk-subtilisin and degraded by Tk-subtilisin to release active Tk-subtilisin. To examine whether the mutation of Leu69 to Pro, which is the most unfavourable residue in the P1 position for subtilisins, affects the maturation of Pro-Tk-subtilisin, the Pro-Tk-subtilisin and Tk-propeptide derivatives with this mutation (Pro-L69P and L69P-propeptide) were constructed and characterized. Pro-L69P was autoprocessed more slowly than Pro-Tk-subtilisin. Nevertheless, it matured to Tk-subtilisin more rapidly than Pro-Tk-subtilisin because L69P-propeptide was degraded by Tk-subtilisin more rapidly than Tk-propeptide. The chaperone function and stability of L69P-propeptide were comparable to those of Tk-propeptide, whereas the inhibitory potency and binding ability of L69P-propeptide were considerably reduced compared to those of Tk-propeptide. The crystal structure of the complex between L69P-propeptide and S324A-subtilisin (i.e. a protease activity-defective mutant) revealed that the C-terminal region of L69P-propeptide does not well fit into the substrate binding pockets of Tk-subtilisin (S1-S4 subsites) as a result of a conformational change caused by the mutation. These results suggest that the Leu?Pro mutation accelerates the maturation of Pro-Tk-subtilisin by reducing the binding ability of Tk-propeptide to Tk-subtilisin.
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Isolation of a novel cutinase homolog with polyethylene terephthalate-degrading activity from leaf-branch compost by using a metagenomic approach.
Appl. Environ. Microbiol.
PUBLISHED: 12-22-2011
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The gene encoding a cutinase homolog, LC-cutinase, was cloned from a fosmid library of a leaf-branch compost metagenome by functional screening using tributyrin agar plates. LC-cutinase shows the highest amino acid sequence identity of 59.7% to Thermomonospora curvata lipase. It also shows the 57.4% identity to Thermobifida fusca cutinase. When LC-cutinase without a putative signal peptide was secreted to the periplasm of Escherichia coli cells with the assistance of the pelB leader sequence, more than 50% of the recombinant protein, termed LC-cutinase*, was excreted into the extracellular medium. It was purified and characterized. LC-cutinase* hydrolyzed various fatty acid monoesters with acyl chain lengths of 2 to 18, with a preference for short-chain substrates (C(4) substrate at most) most optimally at pH 8.5 and 50°C, but could not hydrolyze olive oil. It lost activity with half-lives of 40 min at 70°C and 7 min at 80°C. LC-cutinase* had an ability to degrade poly(?-caprolactone) and polyethylene terephthalate (PET). The specific PET-degrading activity of LC-cutinase* was determined to be 12 mg/h/mg of enzyme (2.7 mg/h/?kat of pNP-butyrate-degrading activity) at pH 8.0 and 50°C. This activity is higher than those of the bacterial and fungal cutinases reported thus far, suggesting that LC-cutinase* not only serves as a good model for understanding the molecular mechanism of PET-degrading enzyme but also is potentially applicable for surface modification and degradation of PET.
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Flavobacterium compostarboris sp. nov., isolated from leaf-and-branch compost, and emended descriptions of Flavobacterium hercynium, Flavobacterium resistens and Flavobacterium johnsoniae.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 10-21-2011
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A strictly aerobic, Gram-negative, yellow-pigmented, non-spore-forming rod, designated 15C3(T), was isolated from aerobic leaf-and-branch compost at EXPO Park in Osaka, Japan. Growth was observed at 9-33 °C (optimum 25 °C) and pH 5.6-7.9 (optimum pH 6.1-7.0). No growth occurred with >2% (w/v) NaCl. Strain 15C3(T) reduced nitrate to nitrogen and showed catalase activity but not oxidase activity. The predominant fatty acids were iso-C(15:0), iso-C(17:0) 3-OH and summed feature 3 (comprising C(16:1)?7c and/or iso-C(15:0) 2-OH). The isolate contained phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone. The G+C content of the genomic DNA of strain 15C3(T) was 33.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15C3(T) belonged to the genus Flavobacterium and was most closely related to Flavobacterium hercynium WB 4.2-33(T) (96.9% sequence similarity). On the basis of phenotypic and phylogenetic distinctiveness, strain 15C3(T) is considered to represent a novel species in the genus Flavobacterium, for which the name Flavobacterium compostarboris sp. nov. is proposed. The type strain is 15C3(T) (?=?KACC 14224(T) ?=?JCM 16527(T)). Emended descriptions of F. hercynium, Flavobacterium resistens and Flavobacterium johnsoniae are also given.
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Crystal structure of N-domain of FKBP22 from Shewanella sp. SIB1: dimer dissociation by disruption of Val-Leu knot.
Protein Sci.
PUBLISHED: 06-09-2011
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FK506-binding protein 22 (FKBP22) from the psychrotophic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. Each monomer consists of the N-terminal domain responsible for dimerization and C-terminal catalytic domain. To reveal interactions at the dimer interface of SIB1 FKBP22, the crystal structure of the N-domain of SIB1 FKBP22 (SN-FKBP22, residues 1-68) was determined at 1.9 Å resolution. SN-FKBP22 forms a dimer, in which each monomer consists of three helices (?1, ?2, and ?3N). In the dimer, two monomers have head-to-head interactions, in which residues 8-64 of one monomer form tight interface with the corresponding residues of the other. The interface is featured by the presence of a Val-Leu knot, in which Val37 and Leu41 of one monomer interact with Val41 and Leu37 of the other, respectively. To examine whether SIB1 FKBP22 is dissociated into the monomers by disruption of this knot, the mutant protein V37R/L41R-FKBP22, in which Val37 and Leu41 of SIB1 FKBP22 are simultaneously replaced by Arg, was constructed and biochemically characterized. This mutant protein was indistinguishable from the SIB1 FKBP22 derivative lacking the N-domain in oligomeric state, far-UV CD spectrum, thermal denaturation curve, PPIase activity, and binding ability to a folding intermediate of protein, suggesting that the N-domain of V37R/L41R-FKBP22 is disordered. We propose that a Val-Leu knot at the dimer interface of SIB1 FKBP22 is important for dimerization and dimerization is required for folding of the N-domain.
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FK506-Binding Protein 22 from a Psychrophilic Bacterium, a Cold Shock-Inducible Peptidyl Prolyl Isomerase with the Ability to Assist in Protein Folding.
Int J Mol Sci
PUBLISHED: 05-12-2011
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Adaptation of microorganisms to low temperatures remains to be fully elucidated. It has been previously reported that peptidyl prolyl cis-trans isomerases (PPIases) are involved in cold adaptation of various microorganisms whether they are hyperthermophiles, mesophiles or phsycrophiles. The rate of cis-trans isomerization at low temperatures is much slower than that at higher temperatures and may cause problems in protein folding. However, the mechanisms by which PPIases are involved in cold adaptation remain unclear. Here we used FK506-binding protein 22, a cold shock protein from the psychrophilic bacterium Shewanella sp. SIB1 (SIB1 FKBP22) as a model protein to decipher the involvement of PPIases in cold adaptation. SIB1 FKBP22 is homodimer that assumes a V-shaped structure based on a tertiary model. Each monomer consists of an N-domain responsible for dimerization and a C-catalytic domain. SIB1 FKBP22 is a typical cold-adapted enzyme as indicated by the increase of catalytic efficiency at low temperatures, the downward shift in optimal temperature of activity and the reduction in the conformational stability. SIB1 FKBP22 is considered as foldase and chaperone based on its ability to catalyze refolding of a cis-proline containing protein and bind to a folding intermediate protein, respectively. The foldase and chaperone activites of SIB1 FKBP22 are thought to be important for cold adaptation of Shewanella sp. SIB1. These activities are also employed by other PPIases for being involved in cold adaptation of various microorganisms. Despite other biological roles of PPIases, we proposed that foldase and chaperone activities of PPIases are the main requirement for overcoming the cold-stress problem in microorganisms due to folding of proteins.
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Identification of the substrate binding site in the N-terminal TBP-like domain of RNase H3.
FEBS Lett.
PUBLISHED: 04-26-2011
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Ribonuclease H3 from Bacillus stearothermophilus (Bst-RNase H3) has the N-terminal TBP-like substrate-binding domain. To identify the substrate binding site in this domain, the mutant proteins of the intact protein and isolated N-domain, in which six of the seventeen residues corresponding to those involved in DNA binding of TBP are individually mutated to Ala, were constructed. All of them exhibited decreased enzymatic activities and/or substrate-binding affinities when compared to those of the parent proteins, suggesting that the N-terminal domain of RNase H3 uses the flat surface of the ?-sheet for substrate binding as TBP to bind DNA. This domain may greatly change conformation upon substrate binding.
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An alternative mature form of subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis identified in the presence of Ca2+.
FEBS J.
PUBLISHED: 04-13-2011
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Pro-Tk-SP from Thermococcus kodakaraensis consists of the four domains: N-propeptide, subtilisin (EC 3.4.21.62) domain, ?-jelly roll domain and C-propeptide. To analyze the maturation process of this protein, the Pro-Tk-SP derivative with the mutation of the active-site serine residue to Cys (Pro-Tk-S359C), Pro-Tk-S359C derivatives lacking the N-propeptide (ProC-Tk-S359C) and both propeptides (Tk-S359C), and a His-tagged form of the isolated C-propeptide (ProC*) were constructed. Pro-Tk-S359C was purified mostly in an autoprocessed form in which the N-propeptide is autoprocessed but the isolated N-propeptide (ProN) forms a stable complex with ProC-Tk-S359C, indicating that the N-propeptide is autoprocessed first. The subsequent maturation process was analyzed using ProC-Tk-S359C, instead of the ProN:ProC-Tk-S359C complex. The C-propeptide was autoprocessed and degraded when ProC-Tk-S359C was incubated at 80 °C in the absence of Ca(2+). However, it was not autoprocessed in the presence of Ca(2+). Comparison of the susceptibility of ProC* to proteolytic degradation in the presence and absence of Ca(2+) suggests that the C-propeptide becomes highly resistant to proteolytic degradation in the presence of Ca(2+). We propose that Pro-Tk-SP derivative lacking N-propeptide (Val114-Gly640) represents a mature form of Pro-Tk-SP in a natural environment. The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T(m) value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9 °C in the absence of Ca(2+) and 7.5 °C in the presence of Ca(2+), indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C.
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Stabilization by fusion to the C-terminus of hyperthermophile Sulfolobus tokodaii RNase HI: a possibility of protein stabilization tag.
PLoS ONE
PUBLISHED: 01-19-2011
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RNase HI from the hyperthermophile Sulfolobus tokodaii (Sto-RNase HI) is stabilized by its C-terminal residues. In this work, the stabilization effect of the Sto-RNase HI C-terminal residues was investigated in detail by thermodynamic measurements of the stability of variants lacking the disulfide bond (C58/145A), or the six C-terminal residues (?C6) and by structural analysis of ?C6. The results showed that the C-terminal does not affect overall structure and stabilization is caused by local interactions of the C-terminal, suggesting that the C-terminal residues could be used as a "stabilization tag." The Sto-RNase HI C-terminal residues (-IGCIILT) were introduced as a tag on three proteins. Each chimeric protein was more stable than its wild-type protein. These results suggested the possibility of a simple stabilization technique using a stabilization tag such as Sto-RNase HI C-terminal residues.
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The N-terminal hybrid binding domain of RNase HI from Thermotoga maritima is important for substrate binding and Mg2+-dependent activity.
FEBS J.
PUBLISHED: 09-28-2010
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Thermotoga maritima ribonuclease H (RNase H) I (Tma-RNase HI) contains a hybrid binding domain (HBD) at the N-terminal region. To analyze the role of this HBD, Tma-RNase HI, Tma-W22A with the single mutation at the HBD, the C-terminal RNase H domain (Tma-CD) and the N-terminal domain containing the HBD (Tma-ND) were overproduced in Escherichia coli, purified and biochemically characterized. Tma-RNase HI prefers Mg(2+) to Mn(2+) for activity, and specifically loses most of the Mg(2+)-dependent activity on removal of the HBD and 87% of it by the mutation at the HBD. Tma-CD lost the ability to suppress the RNase H deficiency of an E. coli rnhA mutant, indicating that the HBD is responsible for in vivo RNase H activity. The cleavage-site specificities of Tma-RNase HI are not significantly changed on removal of the HBD, regardless of the metal cofactor. Binding analyses of the proteins to the substrate using surface plasmon resonance indicate that the binding affinity of Tma-RNase HI is greatly reduced on removal of the HBD or the mutation. These results indicate that there is a correlation between Mg(2+)-dependent activity and substrate binding affinity. Tma-CD was as stable as Tma-RNase HI, indicating that the HBD is not important for stability. The HBD of Tma-RNase HI is important not only for substrate binding, but also for Mg(2+)-dependent activity, probably because the HBD affects the interaction between the substrate and enzyme at the active site, such that the scissile phosphate group of the substrate and the Mg(2+) ion are arranged ideally.
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Highly active metallocarboxypeptidase from newly isolated Geobacillus strain SBS-4S: cloning and characterization.
J. Biosci. Bioeng.
PUBLISHED: 08-27-2010
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The carboxypeptidase gene from Geobacillus SBS-4S was cloned and sequenced. The sequence analysis displayed the gene consists of an open reading frame of 1503 nucleotides encoding a protein of 500 amino acids (CBP(SBS)). The amino acid sequence comparison revealed that CBP(SBS) exhibited a highest homology of 41.6% (identity) with carboxypeptidase Taq from Thermus aquaticus among the characterized proteases. CBP(SBS) contained an active site motif (265)HEXXH(269) which is conserved in family-M32 of carboxypeptidases. The gene was expressed with His-Tag utilizing Escherichia coli expression system and purified to apparent homogeneity. The purified CBP(SBS) showed highest activity at pH 7.5 and 70°C. The enzyme activity was metal ion dependent. Among metal ions highest activity was found in the presence of Co(2+). Thermostability studies of CBP(SBS) by circular dichroism spectroscopy demonstrated the melting temperature of the protein around 77°C. The enzyme exhibited K(m) and V(max) values of 14 mM and 10526 ?mol min(-1) mg(-1) when carbobenzoxy-alanine-arginine was used as substrate. k(cat) and k(cat)/K(m) valves were 10175 s(-1) and 726 mM(-1) s(-1). To our knowledge this is the highest ever reported enzyme activity of a metallocarboxypeptidase and the first characterization of a metallocarboxypeptidase from genus Geobacillus.
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Flavobacterium banpakuense sp. nov., isolated from leaf-and-branch compost.
Int. J. Syst. Evol. Microbiol.
PUBLISHED: 08-06-2010
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A strictly aerobic, Gram-stain-negative, yellow-pigmented, non-spore-forming, motile (by gliding), rod-shaped bacterium, designated strain 15F3(T), was isolated from leaf-and-branch compost. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 15F3(T) was most closely related to Flavobacterium reichenbachii WB 3.2-61(T) and formed a distinct phyletic lineage within the genus Flavobacterium, the type genus of the family Flavobacteriaceae. Growth was observed at 10-34 °C (optimum, 30 °C) and pH 6.0-8.0 (optimum, pH 7.0). No growth occurred in the presence of ?2?% (w/v) NaCl. Strain 15F3(T) reduced nitrate to nitrogen and showed catalase activity but no oxidase activity. The predominant cellular fatty acids were iso-C(15?:?0) and summed feature 3 (comprising C(16?:?1)?7c and/or iso-C(15?:?0) 2-OH). The major isoprenoid quinone was menaquinone-6. The G+C content of the genomic DNA was 31.1 mol%. On the basis of data from this polyphasic study, strain 15F3(T) may be classified as a representative of a novel species within the genus Flavobacterium, for which the name Flavobacterium banpakuense sp. nov. is proposed; the type strain is 15F3(T) (?=?KACC 14225(T) ?=?JCM 16466(T)).
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Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis.
Biochim. Biophys. Acta
PUBLISHED: 06-01-2010
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A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (k(ass)) of 5.2×10³ M?¹ s?¹ at 40°C and 3.1×10? M?¹ s?¹ at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k(ass) values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3-7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.
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Crystal structure of stable protein CutA1 from psychrotrophic bacterium Shewanella sp. SIB1.
J Synchrotron Radiat
PUBLISHED: 04-28-2010
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CutA1 is widely found in bacteria, plants and animals, including humans. The functions of CutA1, however, have not been well clarified. It is known that CutA1s from Pyrococcus horikoshii, Thermus thermophilus and Oryza sativa unfold at temperatures remarkably higher than the growth temperatures of the host organisms. In this work the crystal structure of CutA1 from the psychrotrophic bacterium Shewanella sp. SIB1 (SIB1-CutA1) in a trimeric form was determined at 2.7?Å resolution. This is the first crystal structure of a psychrotrophic CutA1. The overall structure of SIB1-CutA1 is similar to those of CutA1 from Homo sapiens, Escherichia coli, Pyrococcus horikoshii, Thermus thermophilus, Termotoga maritima, Oryza sativa and Rattus norvergicus. A peculiarity is observed in the ?2 strand. The ?2 strand is divided into two short ? strands, ?2a and ?2b, in SIB1-CutA1. A thermal denaturation experiment revealed that SIB1-CutA1 does not unfold completely at 363?K at pH 7.0, although Shewanella sp. SIB1 cannot grow at temperatures exceeding 303?K. These results indicate that the trimeric structural motif of CutA1 is the critical factor in its unusually high stability and suggest that CutA1 needs to maintain its high stability in order to function, even in psychrotrophs.
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Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.
J. Mol. Biol.
PUBLISHED: 03-31-2010
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Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment.
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Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins.
BMC Evol. Biol.
PUBLISHED: 03-29-2010
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The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).
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Protein core adaptability: crystal structures of the cavity-filling variants of Escherichia coli RNase HI.
Protein Pept. Lett.
PUBLISHED: 02-02-2010
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It has generally been accepted that an increase in protein stability is proportional to the increase in hydrophobicity. When a cavity is created by large-to-small substitutions of amino acid residues in protein cores, protein stability decreases 5.3 kJ/mol per single methyl(ene) group removal. In contrast, many reported cavity-filling mutations either failed to increase stability or produced marginal increases in stability; even in successful cases, the increase in stability was much lower than expected from the cost of single methyl(ene) group removal in cavity-creating mutations. Previously it was found that some cavity-filling mutant proteins at Ala52 in E. coli RNase HI increased stability, but decreased activity and they did not increase the stability to the degree expected by the hydrophobic effect alone. The present study attempted to structurally analyze these variant proteins, and it was found that substitutions have little effect on the overall fold but cause conformational strains with the neighboring residues. The present results and literature on cavity-creating/-filling variants provide insight into protein architecture, indicating that natural protein cores are able to accommodate larger side-chain residue by substitution; in other words, excess-packing may not be chosen in natural selection.
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X-ray crystallographic and MD simulation studies on the mechanism of interfacial activation of a family I.3 lipase with two lids.
J. Mol. Biol.
PUBLISHED: 01-13-2010
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The interfacial activation mechanism of family I.3 lipase from Pseudomonas sp. MIS38 (PML), which has two alpha-helical lids (lid1 and lid2), was investigated using a combination of X-ray crystallography and molecular dynamics (MD) simulation. The crystal structure of PML in an open conformation was determined at 2.1 A resolution in the presence of Ca(2+) and Triton X-100. Comparison of this structure with that in the closed conformation indicates that both lids greatly change their positions and lid1 is anchored by the calcium ion (Ca1) in the open conformation. This structure was not seriously changed even when the protein was dialyzed extensively against the Ca(2+)-free buffer containing Triton X-100 before crystallization, indicating that the open conformation is fairly stable unless a micellar substance is removed. The crystal structure of the PML derivative, in which the active site serine residue (Ser207) is diethylphosphorylated by soaking the crystal of PML in the open conformation in a solution containing diethyl p-nitrophenyl phosphate, was also determined. This structure greatly resembles that in the open conformation, indicating that PML structure in the open conformation represents that in the active form. MD simulation of PML in the open conformation in the absence of micelles showed that lid2 closes first, while lid1 maintains its open conformation. Likewise, MD simulation of PML in the closed conformation in the absence of Ca(2+) and in the presence of octane or trilaurin micelles showed that lid1 opens, while lid2 remains closed. These results suggest that Ca1 functions as a hook for stabilization of a fully opened conformation of lid1 and for initiation of subsequent opening of lid2.
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Natural synergism of acid and lactone type mixed sophorolipids in interfacial activities and cytotoxicities.
J Oleo Sci
PUBLISHED: 10-22-2009
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Sophorolipids (SLs) naturally produced from Candida bombicola are a mixture of lactonic (SL-lactone) and acidic (SL-acid) sophorosides of 17-L-hydroxydecanoic acid with an SL-lactone:SL-acid ratio of 72:28. SLs are biodegradable low-foaming surfactants with high detergency and hardness-tolerance properties. To analyze the effect of the SL-lactone:SL-acid ratio on these properties, SL-LXs containing X% SL-lactone, in which X varied from 0 to 100, were prepared and their interfacial activities and cytotoxicities examined. The minimum surface tension values for all SLs examined were comparable. The critical micelle concentration (CMC) was 680 mg/L for SL-L0 and 62-110 mg/L for the other SLs. Interestingly, natural SL (SL-L72) had the lowest surface tension and CMC among all of the SLs examined. The foaming ability and stability of the SLs were dependent on the SL-L content. SL-L0 and L17 had higher foaming values than the other SLs examined in 0-ppm hardness water. These values greatly reduced and became constant when the SL-L content increased over 55%. The detergencies of all of the SLs examined were comparable, except for those of SL-L0 and SL-L100, which were slightly lower than those of the other SLs. These results suggest that natural synergism between SLs creates a better balance for many interfacial activities. The cytotoxicity of SL-L72 was higher than that of SL-L0, but was comparable to that of surfactin, which is commercially available for cosmetic use. The low cytotoxicities and high interfacial properties of SLs increase their usefulness as biocompatible surface active agents for many applications.
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Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.
Biochemistry
PUBLISHED: 10-10-2009
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Tk-subtilisin from the hyperthermophiolic archaeon Thermococcus kodakaraensis matures from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide [Tanaka, S., Saito, K., Chon, H., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S. (2007) J. Biol. Chem. 282, 8246-8255]. It requires Ca(2+) for folding and assumes a molten globule-like structure in the absence of Ca(2+) even in the presence of Tk-propeptide. Tk-subtilisin contains seven Ca(2+)-binding sites. Four of them (Ca2-Ca5) are located within a long loop, which mostly consists of a unique insertion sequence of this protein. To analyze the role of this Ca(2+)-binding loop, three mutant proteins, Deltaloop-Tk-subtilisin, DeltaCa2-Pro-S324A, and DeltaCa3-Pro-S324A, were constructed. These proteins were designed to remove the Ca(2+)-binding loop, Ca2 site, or Ca3 site of Pro-Tk-subtilisin or its active site mutant Pro-S324A. Far-UV CD spectra of these proteins refolded in the absence and presence of Ca(2+) indicated that Deltaloop-Tk-subtilisin completely lost the ability to fold into a native structure. In contrast, two other proteins retained this ability, although their refolding rates were greatly decreased compared to that of Pro-S324A. Determination of the crystal structures of these proteins purified in a Ca(2+)-bound form indicates that the structures of DeltaCa2-Pro-S324A and DeltaCa3-Pro-S324A are virtually identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca(2+)-binding loop is destabilized. Nevertheless, these proteins were slightly more stable than Pro-S324A. These results suggest that the Ca(2+)-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure.
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Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein.
J. Biochem.
PUBLISHED: 07-16-2009
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Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.
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Engineering of monomeric FK506-binding protein 22 with peptidyl prolyl cis-trans isomerase. Importance of a V-shaped dimeric structure for binding to protein substrate.
FEBS J.
PUBLISHED: 06-25-2009
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FK506-binding protein 22 (FKBP22) from the psychrotrophic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) (EC 5.2.1.8) activity. Each monomer consists of 205 amino acid residues. According to a tertiary model, SIB1 FKBP22 assumes a V-shaped structure, in which two monomers interact with each other at their N-termini. Each monomer consists of an N-terminal domain with a dimerization core and a C-terminal catalytic domain, which are separated by a 40-residue-long a-helix. To clarify the role of this V-shaped structure, we constructed a mutant protein, in which the N-domain is tandemly repeated through a flexible linker. This protein, termed NNC-FKBP22, is designed such that two repetitive N-domains are folded into a structure similar to that of the Shewanella sp. SIB1 FKBP22 wild-type protein (WT). NNC-FKBP22 was overproduced in Escherichia coli in a His-tagged form, purified and biochemically characterized. Gel-filtration chromatography and ultracentrifugation analyses indicate that NNC-FKBP22 exists as a monomer. Analysis of thermal denaturation using differential scanning calorimetry indicates that NNC-FKBP22 unfolds with two transitions, as does the WT protein. NNC-FKBP22 exhibited PPIase activity for both peptide and protein substrates. However, in contrast to its activity for peptide substrate, which was comparable to that of the WT protein, its activity for protein substrate was reduced by five- to six-fold, compared to that of the WT. Surface plasmon resonance analyses indicate that NNC-FKBP22 binds to a reduced form of a-lactalbumin with a six-fold weaker affinity than that of WT. These results suggest that a V-shaped structure of SIB1 FKBP22 is important for efficient binding to a protein substrate.
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Identification of the interactions critical for propeptide-catalyzed folding of Tk-subtilisin.
J. Mol. Biol.
PUBLISHED: 06-16-2009
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Tk-subtilisin requires Ca(2+) for folding. This folding is accelerated by the chaperone function of its propeptide (Tkpro). Several Tkpro and Tk-subtilisin derivatives were constructed to examine whether the interactions between the C-terminal extended region of Tkpro and Tk-subtilisin and Glu61/Asp63- and Glu201-mediated hydrogen bonds at the domain interface are important for the chaperone function of Tkpro. The Tkpro derivatives with a series of C-terminal truncations and double mutations at Glu61 and Asp63 exhibited weaker chaperone functions than Tkpro for SA-subtilisin (active-site mutant of Tk-subtilisin). Good correlation was observed between their chaperone functions and binding abilities to the folded SA-subtilisin protein. These results suggest that the C-terminal extended region, Glu61, and Asp63 of Tkpro are not critical for folding of Tk-subtilisin but accelerate it by binding to a folding intermediate of Tk-subtilisin with a native-like structure at their binding sites. In contrast, Tkpro exhibited little chaperone function for E201A/SA-subtilisin. It could bind to the folded E201A/SA-subtilisin protein with a lower association constant than that for SA-subtilisin. These results suggest a loop of Tkpro, which interacts with Glu201 of Tk-subtilisin through hydrogen bonds and is required for folding of Tk-subtilisin by binding to a folding intermediate of Tk-subtilisin with a nonnative structure. Because this loop is fairly hydrophobic and tightly packs to the surface parallel helices of the central alphabetaalpha substructure of Tk-subtilisin, binding of this loop to Glu201 may induce association of these two helices and thereby formation of the alphabetaalpha substructure. We propose that Glu201-mediated interactions are critical for initiation of Tkpro-catalyzed folding of Tk-subtilisin.
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Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23.
Extremophiles
PUBLISHED: 06-08-2009
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Geobacillus thermoleovorans B23 is capable of degrading long-chain alkanes at 70 degrees C. Bt-aldh, an aldehyde dehydrogenase gene in B23, was located in the upstream region of p21 whose expression level was dramatically increased when alkane degradation was started (Kato et al. 2009, BMC Microbiol 9:60). Like p21, transcription level of Bt-aldh was also increased upon alkane degradation. Bt-Aldh (497 aa, MW = 53,886) was overproduced in Escherichia coli, purified, and characterized biochemically. Bt-Aldh acted as an octamer, required NAD(+) as a coenzyme, and showed high activity against aliphatic long-chain aldehydes such as tetradecanal. The optimum condition for activity was 50-55 degrees C and pH 10.0. The activity was elevated to two- to threefold in the presence of 2 mM Ba(2+), Ca(2+), or Sr(2+), while Mg(2+) and Zn(2+) inhibited the enzyme activity. Bt-Aldh represents thermophilic aldehyde dehydrogenases responsible for degradation of long-chain alkanes.
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Alkane inducible proteins in Geobacillus thermoleovorans B23.
BMC Microbiol.
PUBLISHED: 03-25-2009
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Initial step of beta-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution.
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Ribonuclease H: molecular diversities, substrate binding domains, and catalytic mechanism of the prokaryotic enzymes.
FEBS J.
PUBLISHED: 02-18-2009
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The prokaryotic genomes, for which complete nucleotide sequences are available, always contain at least one RNase H gene, indicating that RNase H is ubiquitous in all prokaryotic cells. Coupled with its unique substrate specificity, the enzyme has been expected to play crucial roles in the biochemical processes associated with DNA replication, gene expression and DNA repair. The physiological role of prokaryotic RNases H, especially of type 1 RNases H, has been extensively studied using Escherichia coli strains that are defective in RNase HI activity or overproduce RNase HI. However, it is not fully understood yet. By contrast, significant progress has been made in this decade in identifying novel RNases H with respect to their biochemical properties and structures, and elucidating catalytic mechanism and substrate recognition mechanism of RNase H. We review the results of these studies.
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Destabilization of psychrotrophic RNase HI in a localized fashion as revealed by mutational and X-ray crystallographic analyses.
FEBS J.
PUBLISHED: 01-06-2009
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The Arg97 --> Gly and Asp136 --> His mutations stabilized So-RNase HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 by 5.4 and 9.7 degrees C, respectively, in T(m), and 3.5 and 6.1 kJ x mol(-1), respectively, in DeltaG(H2O). These mutations also stabilized the So-RNase HI derivative (4x-RNase HI) with quadruple thermostabilizing mutations in an additive manner. As a result, the resultant sextuple mutant protein (6x-RNase HI) was more stable than the wild-type protein by 28.8 degrees C in T(m) and 27.0 kJ x mol(-1) in DeltaG(H2O). To analyse the effects of the mutations on the protein structure, the crystal structure of the 6x-RNase HI protein was determined at 2.5 A resolution. The main chain fold and interactions of the side-chains of the 6x-RNase HI protein were basically identical to those of the wild-type protein, except for the mutation sites. These results indicate that all six mutations independently affect the protein structure, and are consistent with the fact that the thermostabilizing effects of the mutations are roughly additive. The introduction of favourable interactions and the elimination of unfavourable interactions by the mutations contribute to the stabilization of the 6x-RNase HI protein. We propose that So-RNase HI is destabilized when compared with its mesophilic and thermophilic counterparts in a localized fashion by increasing the number of amino acid residues unfavourable for protein stability.
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Enhancement of the enzymatic activity of Escherichia coli acetyl esterase by a double mutation obtained by random mutagenesis.
Biosci. Biotechnol. Biochem.
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A double mutant of Escherichia coli acetyl esterase (EcAE) with enhanced enzymatic activity was obtained by random mutagenesis using error-prone PCR and screening for enzymatic activity by observing halo formation on a tributyrin plate. The mutant contained Leu97Phe (L97F) and Leu209Phe (L209F) mutations. Single mutants L97F and L209F were also constructed and analyzed for kinetic parameters, as well as double mutant L97F/L209F. Kinetic analysis using p-nitrophenyl butyrate as substrate indicated that the k(cat) values of L97F and L97F/L209F were larger than that of the wild-type enzyme, by 8.3-fold and 12-fold respectively, whereas no significant change was observed in the k(cat) value of L209F. The K(m) values of L209F and L97F/L209F were smaller than that of the wild-type enzyme, by 2.9-fold and 2.4-fold respectively, whereas no significant change was observed in the K(m) value of L97F. These results indicate that a combination of an increase in k(cat) values due to the L97F mutation and a decrease in K(m) value due to the L209F mutation renders the k(cat)/K(m) value of the double mutant enzyme 29-fold higher than that of the wild-type enzyme.
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Slow unfolding pathway of hyperthermophilic Tk-RNase H2 examined by pulse proteolysis using the stable protease Tk-subtilisin.
Biochemistry
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The unfolding speed of some hyperthermophilic proteins is significantly slower than those of their mesostable homologues. Ribonuclease H2 from the hyperthermophilic archaeon Thermococcus kodakarensis (Tk-RNase H2) is stabilized by its remarkably slow unfolding rate. In this work, we examined the slow unfolding pathway of Tk-RNase H2 by pulse proteolysis using a superstable subtilisin-like serine protease from T. kodakarensis (Tk-subtilisin). Tk-subtilisin has enzymatic activity in highly concentrated guanidine hydrochloride (GdnHCl), in which Tk-RNase H2 unfolds slowly. The native state of Tk-RNase H2 was completely resistant to Tk-subtilisin, whereas the unfolded state (induced by 4 M GdnHCl) was degraded by Tk-subtilisin. Degradation products of Tk-RNase H2 created from pulse proteolysis during its unfolding were detected by tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We identified the cleavage sites in Tk-RNase H2 by N-terminal sequencing and mass spectrometry and constructed mimics of the unfolding intermediate of Tk-RNase H2 by protein engineering. The mimics were biophysically characterized. We found that the native state of Tk-RNase H2 (N-state) changed to the I(A)-state that was digested by Tk-subtilisin in the early stage of unfolding. In the slow unfolding pathway, the I(A)-state shifted to two intermediate forms, I(B)-state and I(C)-state. The I(B)-state was digested by Tk-subtilisin in the C-terminal region, but the I(C)-state was a Tk-subtilisin resistant form. These states gradually unfolded through the I(D)-state, in which the N-terminal region was digested. The results indicate that pulse proteolysis, by a superstable protease, was a suitable strategy and an effective tool for analyzing intermediate structures of proteins with slow unfolding properties. We also showed that the N-terminal region contributes to the slow unfolding of Tk-RNase H2, and the C-terminal region is important for folding and stability.
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Requirement of insertion sequence IS1 for thermal adaptation of Pro-Tk-subtilisin from hyperthermophilic archaeon.
Extremophiles
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Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis matures from Pro-Tk-subtilisin (Pro-TKS) upon autoprocessing and degradation of propeptide. Pro-TKS contains the insertion sequence (IS1) at the N-terminus of the mature domain as compared to bacterial pro-subtilisins. To analyze the role of IS1, the Pro-TKS derivative without IS1 (?IS1-Pro-TKS) and its active-site mutants (?IS1-Pro-S324A and ?IS1-Pro-S324C) were constructed and characterized. ?IS1-Pro-S324A and ?IS1-Pro-TKS represent an unautoprocessed and autoprocessed form of ?IS1-Pro-TKS, respectively. The CD and ANS fluorescence spectra of these proteins indicate that folding of ?IS1-Pro-TKS is not completed by binding of Ca(2+) ions but is completed by the subsequent autoprocessing reaction. Thermal denaturation of these proteins analyzed by DSC and CD spectroscopy indicates that unautoprocessed ?IS1-Pro-TKS is less stable than autoprocessed ?IS1-Pro-TKS by 26.3 °C in T (m). The stability of autoprocessed ?IS1-Pro-TKS is comparable to that of Pro-TKS, which is slightly lower than that of unautoprocessed Pro-TKS. These results suggest that ?IS1-Pro-TKS is fully folded and greatly stabilized by autoprocessing. ?IS1-Pro-TKS more slowly matured to ?IS1-Tk-subtilisin than Pro-TKS did, due to a decrease in the autoprocessing rate. We propose that IS1 is required not only for hyperstabilization of Pro-TKS but also for its rapid maturation.
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Requirement of lid2 for interfacial activation of a family I.3 lipase with unique two lid structures.
FEBS J.
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A family I.3 lipase from Pseudomonas sp. MIS38 (PML) is characterized by the presence of two lids (lid1 and lid2) that greatly change conformation upon substrate binding. While lid1 represents the commonly known lid in lipases, lid2 is unique to PML and other family I.3 lipases. To clarify the role of lid2 in PML, a lid2 deletion mutant (?L2-PML) was constructed by deleting residues 35-64 of PML. ?L2-PML requires calcium ions for both lipase and esterase activities as does PML, suggesting that it exhibits activity only when lid1 is fully open and anchored by the catalytically essential calcium ion, as does PML. However, when the enzymatic activity was determined using triacetin, the activity of PML exponentially increased as the substrate concentration reached and increased beyond the critical micellar concentration, while that of ?L2-PML did not. These results indicate that PML undergoes interfacial activation, while ?L2-PML does not. The activities of ?L2-PML for long-chain triglycerides significantly decreased while its activity for fatty acid ethyl esters increased, compared with those of PML. Comparison of the tertiary models of ?L2-PML in a closed and open conformation, which are optimized by molecular dynamics simulation, with the crystal structures of PML suggests that the hydrophobic surface area provided by lid1 and lid2 in an open conformation is considerably decreased by the deletion of lid2. We propose that the hydrophobic surface area provided by these lids is necessary to hold the micellar substrates firmly to the active site and therefore lid2 is required for interfacial activation of PML.
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Characteristic features of kynurenine aminotransferase allosterically regulated by (alpha)-ketoglutarate in cooperation with kynurenine.
PLoS ONE
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Kynurenine aminotransferase from Pyrococcus horikoshii OT3 (PhKAT), which is a homodimeric protein, catalyzes the conversion of kynurenine (KYN) to kynurenic acid (KYNA). We analyzed the transaminase reaction mechanisms of this protein with pyridoxal-5-phosphate (PLP), KYN and ?-ketoglutaric acid (2OG) or oxaloacetic acid (OXA). 2OG significantly inhibited KAT activities in kinetic analyses, suggesting that a KYNA biosynthesis is allosterically regulated by 2OG. Its inhibitions evidently were unlocked by KYN. 2OG and KYN functioned as an inhibitor and activator in response to changes in the concentrations of KYN and 2OG, respectively. The affinities of one subunit for PLP or 2OG were different from that of the other subunit, as confirmed by spectrophotometry and isothermal titration calorimetry, suggesting that the difference of affinities between subunits might play a role in regulations of the KAT reaction. Moreover, we identified two active and allosteric sites in the crystal structure of PhKAT-2OG complexes. The crystal structure of PhKAT in complex with four 2OGs demonstrates that two 2OGs in allosteric sites are effector molecules which inhibit the KYNA productions. Thus, the combined data lead to the conclusion that PhKAT probably is regulated by allosteric control machineries, with 2OG as the allosteric inhibitor.
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Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii.
FEBS J.
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The hormone-sensitive lipase (HSL) family is comprised of carboxylesterases and lipases with similarity to mammalian HSL. Thermophilic enzymes of this family have a high potential for use in biocatalysis. We prepared and crystallized a carboxylesterase of the HSL family from Sulfolobus tokodaii (Sto-Est), and determined its structures in the presence and absence of an inhibitor. Sto-Est forms a dimer in solution and the crystal structure suggests the presence of a stable biological dimer. We identified a residue close to the dimer interface, R267, which is conserved in archaeal enzymes of HSL family and is in close proximity to the same residue from the other monomer. Mutations of R267 to Glu, Gly and Lys were conducted and the resultant R267 mutants were characterized and crystallized. The structures of R267E, R267G and R267K are highly similar to that of Sto-Est with only slight differences in atomic coordinates. The dimerized states of R267E and R267G are unstable under denaturing conditions or at high temperature, as shown by a urea-induced dimer dissociation experiment and molecular dynamics simulation. R267E is the most unstable mutant protein, followed by R267G and R267K, as shown by the thermal denaturation curve and optimum temperature for activity. From the data, we discuss the importance of R267 in maintaining the dimer integrity of Sto-Est.
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Structure and characterization of RNase H3 from Aquifex aeolicus.
FEBS J.
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The crystal structure of ribonuclease?H3 from Aquifex?aeolicus (Aae-RNase?H3) was determined at 2.0?Å resolution. Aae-RNase?H3 consists of an N-terminal TATA box-binding protein (TBP)-like domain (N-domain) and a C-terminal RNase?H domain (C-domain). The structure of the C-domain highly resembles that of Bacillus?stearothermophilus RNase?H3 (Bst-RNase?H3), except that it contains three disulfide bonds, and the fourth conserved glutamate residue of the Asp-Glu-Asp-Glu active site motif (Glu198) is located far from the active site. These disulfide bonds were shown to contribute to hyper-stabilization of the protein. Non-conserved Glu194 was identified as the fourth active site residue. The structure of the N-domain without the C-domain also highly resembles that of Bst-RNase?H3. However, the arrangement of the N-domain relative to the C-domain greatly varies for these proteins because of the difference in the linker size between the domains. The linker of Bst-RNase?H3 is relatively long and flexible, while that of Aae-RNase?H3 is short and assumes a helix formation. Biochemical characterizations of Aae-RNase?H3 and its derivatives without the N- or C-domain or with a mutation in the N-domain indicate that the N-domain of Aae-RNase?H3 is important for substrate binding, and uses the flat surface of the ?-sheet for substrate binding. However, this surface is located far from the active site and on the opposite side to the active site. We propose that the N-domain of Aae-RNase?H3 is required for initial contact with the substrate. The resulting complex may be rearranged such that only the C-domain forms a complex with the substrate.
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Requirement of Ca(2+) ions for the hyperthermostability of Tk-subtilisin from Thermococcus kodakarensis.
Biochemistry
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Tk-subtilisin, a hyperthermostable subtilisin-like serine protease from Thermococcus kodakarensis, matures from the inactive precursor, Pro-Tk-subtilisin (Pro-TKS), upon autoprocessing and degradation of the propeptide (Tkpro). It contains seven Ca(2+) ions. Four of them (Ca2-Ca5) are responsible for folding of Tk-subtilisin. In this study, to clarify the role of the other three Ca(2+) ions (Ca1, Ca6, and Ca7), we constructed Pro-TKS derivatives lacking the Ca1 ion (Pro-TKS/?Ca1), Ca6 ion (Pro-TKS/?Ca6), and Ca7 ion (Pro-TKS/?Ca7), and their active site mutants (Pro-S324A/?Ca1, Pro-S324A/?Ca6, and Pro-S324A/?Ca7, respectively). Pro-TKS/?Ca6 and Pro-TKS/?Ca7 fully matured into their active forms upon incubation at 80 °C for 30 min as did Pro-TKS. The mature enzymes were as active as Tk-subtilisin at 80 °C, indicating that the Ca6 and Ca7 ions are not important for activity. In contrast, Pro-TKS/?Ca1 matured poorly at 80 °C because of the instability of its mature domain. The enzymatic activity of Tk-subtilisin/?Ca1 was determined to be 50% of that of Tk-subtilisin using the refolded protein. This result suggests that the Ca1 ion is required for the maximal activity of Tk-subtilisin. The refolding rates of all Pro-S324A derivatives were comparable to that of Pro-S324A (active site mutant of Pro-TKS), indicating that these Ca(2+) ions are not needed for folding of Tk-subtilisin. The stabilities of Pro-S324A/?Ca1 and Pro-S324A/?Ca6 were decreased by 26.6 and 11.7 °C, respectively, in T(m) compared to that of Pro-S324A. The half-lives of Tk-subtilisin/?Ca6 and Tk-subtilisin/?Ca7 at 95 °C were 8- and 4-fold lower than that of Tk-subtilisin, respectively. These results suggest that the Ca1, Ca6, and Ca7 ions, especially the Ca1 ion, contribute to the hyperthermostabilization of Tk-subtilisin.
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Activity, stability, and structure of metagenome-derived LC11-RNase H1, a homolog of Sulfolobus tokodaii RNase H1.
Protein Sci.
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Metagenome-derived LC11-RNase H1 is a homolog of Sulfolobus tokodaii RNase H1 (Sto-RNase H1). It lacks a C-terminal tail, which is responsible for hyperstabilization of Sto-RNase H1. Sto-RNase H1 is characterized by its ability to cleave not only an RNA/DNA hybrid but also a double-stranded RNA (dsRNA). To examine whether LC11-RNase H1 also exhibits both RNase H and dsRNase activities, LC11-RNase H1 was overproduced in Escherichia coli, purified, and characterized. LC11-RNase H1 exhibited RNase H activity with similar metal ion preference, optimum pH, and cleavage mode of substrate with those of Sto-RNase H1. However, LC11-RNase H1 did not exhibit dsRNase activity at any condition examined. LC11-RNase H1 was less stable than Sto-RNases H1 and its derivative lacking the C-terminal tail (Sto-RNase H1?C6) by 37 and 13 °C in T(m) , respectively. To understand the structural bases for these differences, the crystal structure of LC11-RNase H1 was determined at 1.4 Å resolution. The LC11-RNase H1 structure is highly similar to the Sto-RNase H1 structure. However, LC11-RNase H1 has two grooves on protein surface, one containing the active site and the other containing DNA-phosphate binding pocket, while Sto-RNase H1 has one groove containing the active site. In addition, LC11-RNase H1 contains more cavities and buried charged residues than Sto-RNase H1. We propose that LC11-RNase H1 does not exhibit dsRNase activity because dsRNA cannot fit to the two grooves on protein surface and that LC11-RNase H1 is less stable than Sto-RNase H1?C6 because of the increase in cavity volume and number of buried charged residues.
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Role of polar and nonpolar residues at the active site for PPIase activity of FKBP22 from Shewanella sp. SIB1.
FEBS J.
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FKBP22 from the psychotropic bacterium Shewanella sp. SIB1 is a homodimeric protein with peptidyl prolyl cis-trans isomerase (PPIase) activity. According to a tertiary model, several nonpolar residues including Trp157 and Phe197 form a substrate-binding cavity, and Asp137 and Arg142, which form a salt bridge, are located at the edge of this cavity. To analyze the role of these residues, nine single (D137A, R142A, W157A/F/Y, F197A/L/Y/W) and one double (D137A/R142A) mutant protein of SIB1 FKBP22 were constructed. The far- and near-UV CD spectra of these mutant proteins suggest that the mutations at Asp137 and Arg142 do not seriously affect the protein structure, while those at Trp157 and Phe197 cause a local conformational change around the mutation site. Each mutation decreased the PPIase activities of SIB1 FKBP22 for peptide and protein substrates similarly without seriously affecting chaperone function. This result indicates that SIB1 FKBP22 does not require PPIase activity for chaperone function. The PPIase activities of R142A, D137A and D137A/R142A decreased in this order, suggesting that Asp137 and Arg142 play a principal and auxiliary role in catalytic function, respectively, but Arg142 can function as a substitute of Asp137. Because the PPIase activity of SIB1 FKBP22 was not fully lost by the removal of all polar residues around the active site, the desolvation effect may also contribute to the enzymatic activity. However, the mutations of Trp157 to Phe or Phe197 to Leu greatly decrease the enzymatic activity, suggesting that the shape of the substrate-binding cavity is also important for enzymatic activity.
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