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Hot Biological Catalysis: Isothermal Titration Calorimetry to Characterize Enzymatic Reactions

Isothermal titration calorimetry (ITC) is a well-described technique that measures the heat released or absorbed during a chemical reaction, using it as ...

University of Bologna

Gasification of solid waste for energy has significant potential given an abundant feed supply and strong policy drivers. Nonetheless, significant ambiguities in the knowledge base are apparent. Consequently this study investigates sulphur mechanisms within a novel two stage fluid bed-plasma gasification process. This paper includes a detailed review of gasification and plasma fundamentals in relation to the specific process, along with insight on MSW based feedstock properties and sulphur pollutant therein. As a first step to understanding sulphur partitioning and speciation within the process, thermodynamic modelling of the fluid bed stage has been performed. Preliminary findings, supported by plant experience, indicate the prominence of solid phase sulphur species (as opposed to H(2)S) - Na and K based species in particular. Work is underway to further investigate and validate this.

Two stage fluid bed-plasma gasification process for solid waste valorisation: technical review and preliminary thermodynamic modelling of sulphur emissions.

Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-Å resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.

The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design.

Helicobacter pylori UreF (HpUreF) is involved in the insertion of Ni(2+) in the urease active site. The recombinant protein in solution is a dimer characterized by an extensive α-helical structure and a well-folded tertiary structure. HpUreF binds two Ni(2+) ions per dimer, with a micromolar dissociation constant, as shown by calorimetry. X-ray absorption spectroscopy indicated that the Ni(2+) ions reside in a five-coordinate pyramidal geometry comprising exclusively N/O-donor ligands derived from the protein, including one or two histidine imidazole and carboxylate ligands. Binding of Ni(2+) does not affect the solution properties of the protein. Mutation to alanine of His229 and/or Cys231, a pair of residues located on the protein surface that interact with H. pylori UreD, altered the affinity of the protein for Ni(2+). This result, complemented by the findings from X-ray absorption spectroscopy, indicates that the Ni(2+) binding site involves His229, and that Cys231 has an indirect structural role in metal binding. An in vivo assay of urease activation demonstrated that H229A HpUreF, C231A HpUreF, and H229/C231 HpUreF are significantly less competent in this process, suggesting a role for a Ni(2+) complex with UreF in urease maturation. This hypothesis was supported by calculations revealing the presence of a tunnel that joins the Cys-Pro-His metal binding site on UreG and an opening on the UreD surface, passing through UreF close to His229 and Cys231, in the structure of the H. pylori UreDFG complex. This tunnel could be used to transfer nickel into the urease active site during apoenzyme-to-holoenzyme activation.

Article	Year
Two stage fluid bed-plasma gasification process for solid waste valorisation: technical review and preliminary thermodynamic modelling of sulphur emissions. Waste management (New York, N.Y.)\| PubMed ID: 21982278	2012
The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry\| PubMed ID: 23412551	2013
Nickel binding properties of Helicobacter pylori UreF, an accessory protein in the nickel-based activation of urease. Journal of biological inorganic chemistry : JBIC : a publication of the Society of Biological Inorganic Chemistry\| PubMed ID: 24292245	2014

Luca Mazzei

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