determination-protein-ligand-interactions-using-differential-scanning

Determination of Protein-ligand Interactions Using Differential Scanning Fluorimetry

A wide range of methods are currently available for determining the dissociation constant between a protein and interacting small molecules. However, most ...

University of Exeter

The putative L-haloacid dehalogenase gene (DehRhb) from a marine Rhodobacteraceae family was cloned and overexpressed in Escherichia coli. The DehRhb protein was shown to be an L-haloacid dehalogenase with highest activity towards brominated substrates with short carbon chains (≤ C3). The optimal temperature for enzyme activity was 55 °C, and the Vmax and Km were 1.75 μm·min(-1) ·mg(-1) of protein and 6.72 mm, respectively, when using monobromoacetic acid as a substrate. DehRhb showed moderate thermal stability, with a melting temperature of 67 °C. The enzyme demonstrated high tolerance to solvents, as shown by thermal shift experiments and solvent incubation assays. The DehRhb protein was crystallized and structures of the native, reaction intermediate and substrate-bound forms were determined. The active site of DehRhb had significant differences from previously studied L-haloacid dehalogenases. The asparagine and arginine residues shown to be essential for catalytic activity in other L-haloacid dehalogenases are not present in DehRhb. The histidine residue which replaces the asparagine residue in DehRhb was coordinated by a conformationally strained glutamate residue that replaces a conserved glycine. The His/Glu dyad is positioned for deprotonation of the catalytic water which attacks the ester bond in the reaction intermediate. The catalytic water in DehRhb is shifted by ~ 1.5 Å from its position in other L-haloacid dehalogenases. A similar His/Glu or Asp dyad is known to activate the catalytic water in haloalkane dehalogenases. The DehRhb enzyme represents a novel member within the L-haloacid dehalogenase family and it has potential to be used as a commercial biocatalyst.

Marine Rhodobacteraceae L-haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water.

The recombinant L-haloacid dehalogenase from the marine bacterium Psychromonas ingrahamii has been cloned and over-expressed in Escherichia coli. It shows activity towards monobromoacetic (100 %), monochloroacetic acid (62 %), S-chloropropionic acid (42 %), S-bromopropionic acid (31 %), dichloroacetic acid (28 %) and 2-chlorobutyric acid (10 %), respectively. The L-haloacid dehalogenase has highest activity towards substrates with shorter carbon chain lengths (≤ C3), without preference towards a chlorine or bromine at the α-carbon position. Despite being isolated from a psychrophilic bacterium, the enzyme has mesophilic properties with an optimal temperature for activity of 45 °C. It retains above 70 % of its activity after being incubated at 65 °C for 90 min before being assayed at 25 °C. The enzyme is relatively stable in organic solvents as demonstrated by activity and thermal shift analysis. The V max and K m were calculated to be 0.6 μM min(-1) mg(-1) and 1.36 mM with monobromoacetic acid, respectively. This solvent-resistant and stable L-haloacid dehalogenase from P. ingrahamii has potential to be used as a biocatalyst in industrial processes.

Characterisation of an L-haloacid dehalogenase from the marine psychrophile Psychromonas ingrahamii with potential industrial application.

A putative haloalkane dehalogenase has been identified in a marine Rhodobacteraceae and subsequently cloned and over-expressed in Escherichia coli. The enzyme has highest activity towards the substrates 1,6-dichlorohexane, 1-bromooctane, 1,3-dibromopropane and 1-bromohexane. The crystal structures of the enzyme in the native and product bound forms reveal a large hydrophobic active site cavity. A deeper substrate binding pocket defines the enzyme preference towards substrates with longer carbon chains. Arg136 at the bottom of the substrate pocket is positioned to bind the distal halogen group of extended di-halogenated substrates.

Article	Year
Marine Rhodobacteraceae L-haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water. The FEBS journal\| PubMed ID: 23384397	2013
Characterisation of an L-haloacid dehalogenase from the marine psychrophile Psychromonas ingrahamii with potential industrial application. Marine biotechnology (New York, N.Y.)\| PubMed ID: 23949008	2013
Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae. FEBS letters\| PubMed ID: 24613925	2014

Halina R. Novak

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