Christopher Sayer

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

The X-ray structures of two ω-aminotransferases from P. aeruginosa and C. violaceum in complex with an inhibitor offer the first detailed insight into the structural basis of the substrate specificity of these industrially important enzymes.

The substrate specificity, enantioselectivity and structure of the (R)-selective amine : pyruvate transaminase from Nectria haematococca

During the last decade the use of transaminases for the production of pharmaceutical and fine chemical intermediates has attracted a great deal of attention. Transaminases are versatile biocatalysts for the efficient production of amine intermediates and many have (S)‐enantiospecificity. Transaminases with (R)‐specificity are needed to expand the applications of these enzymes in biocatalysis. In this work we have identified a fungal putative (R)‐specific transaminase from the Eurotiomycetes Nectria haematococca, cloned a synthetic version of this gene, demonstrated (R)‐selective deamination of several substrates including (R)‐α‐methylbenzylamine, as well as production of (R)‐amines, and determined its crystal structure. The crystal structures of the holoenzyme and the complex with an inhibitor gabaculine offer the first detailed insight into the structural basis for substrate specificity and enantioselectivity of the industrially important class of (R)‐selective amine : pyruvate transaminases.

Discovery and Characterization of a Thermostable and Highly Halotolerant GH5 Cellulase from an Icelandic Hot Spring Isolate.

With the ultimate goal of identifying robust cellulases for industrial biocatalytic conversions, we have isolated and characterized a new thermostable and very halotolerant GH5 cellulase. This new enzyme, termed CelDZ1, was identified by bioinformatic analysis from the genome of a polysaccharide-enrichment culture isolate, initiated from material collected from an Icelandic hot spring. Biochemical characterization of CelDZ1 revealed that it is a glycoside hydrolase with optimal activity at 70°C and pH 5.0 that exhibits good thermostability, high halotolerance at near-saturating salt concentrations, and resistance towards metal ions and other denaturing agents. X-ray crystallography of the new enzyme showed that CelDZ1 is the first reported cellulase structure that lacks the defined sugar-binding 2 subsite and revealed structural features which provide potential explanations of its biochemical characteristics.

The structure of a tetrameric α-carbonic anhydrase from Thermovibrio ammonificans reveals a core formed around intermolecular disulfides that contribute to its thermostability

Carbonic anhydrase enzymes catalyse the reversible hydration of carbon dioxide to bicarbonate. A thermophilic Thermovibrio ammonificans α-carbonic anhydrase (TaCA) has been expressed in Escherichia coli and structurally and biochemically characterized. The crystal structure of TaCA has been determined in its native form and in two complexes with bound inhibitors. The tetrameric enzyme is stabilized by a unique core in the centre of the molecule formed by two intersubunit disulfides and a single lysine residue from each monomer that is involved in intersubunit ionic interactions. The structure of this core protects the intersubunit disulfides from reduction, whereas the conserved intrasubunit disulfides are not formed in the reducing environment of the E. coli host cytosol. When oxidized to mimic the environment of the periplasmic space, TaCA has increased thermostability, retaining 90% activity after incubation at 70°C for 1 h, making it a good candidate for industrial carbon-dioxide capture. The reduction of all TaCA cysteines resulted in dissociation of the tetrameric molecule into monomers with lower activity and reduced thermostability. Unlike other characterized α-carbonic anhydrases, TaCA does not display esterase activity towards p-nitrophenyl acetate, which appears to result from the increased rigidity of its protein scaffold.

Marine Rhodobacteraceae l‐haloacid dehalogenase contains a novel His/Glu dyad that could activate the catalytic water

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.

Discovery and characterization of thermophilic limonene-1,2-epoxide hydrolases from hot spring metagenomic libraries.

The epoxide hydrolases (EHs) represent an attractive option for the synthesis of chiral epoxides and 1,2‐diols which are valuable building blocks for the synthesis of several pharmaceutical compounds. A metagenomic approach has been used to identify two new members of the atypical EH limonene‐1,2‐epoxide hydrolase (LEH) family of enzymes. These two LEHs (Tomsk‐LEH and CH55‐LEH) show EH activities towards different epoxide substrates, differing in most cases from those previously identified for Rhodococcus erythropolis (Re‐LEH) in terms of stereoselectivity. Tomsk‐LEH and CH55‐LEH, both from thermophilic sources, have higher optimal temperatures and apparent melting temperatures than Re‐LEH. The new LEH enzymes have been crystallized and their structures solved to high resolution in the native form and in complex with the inhibitor valpromide for Tomsk‐LEH and poly(ethylene glycol) for CH55‐LEH. The structural analysis has provided insights into the LEH mechanism, substrate specificity and stereoselectivity of these new LEH enzymes, which has been supported by mutagenesis studies.

Biochemical and structural characterisation of a haloalkane dehalogenase from a marine Rhodobacteraceae.

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.

Characterization of a phosphotriesterase-like lactonase from the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia

The phosphotriesterase-like lactonase (PLL) encoded by Vmut_2255 in the hyperthermoacidophilic crenarchaeon Vulcanisaeta moutnovskia (VmutPLL), represents the only hyperthermophilic PLL homologue identified so far in addition to the previously characterized thermophilic PLLs from Sulfolobus spp. The Vmut_2255 gene was cloned, heterologously expressed in Escherichia coli; the resultant protein purified and characterized as a 82kDa homodimer (36kDa subunits). The VmutPLL converted lactones and acyl-homoserine lactones (AHLs) with comparable activities. Towards organophosphates (OP) VmutPLL showed a promiscuous but significantly lower activity and only minor activity was observed with carboxylesters. The catalytic activity strictly depended on bivalent cations (Cd(2+)>Ni(2+)>Co(2+)>Mn(2+)>Zn(2+)). Furthermore, VmutPLL showed a pH optimum around 8.0, a temperature optimum of 80°C, and thermostability with a half-life of 26min at 90°C. In this work, the stereoselectivity of a PLL enzyme was investigated for the first time using enantiopure lactones. The VmutPLL showed a slight preference but not an exclusive specificity for the (R)-enantiomers of capro- and valerolactone. The high thermal stability as well as the broad substrate spectrum towards lactones, AHLs and OPs underlines the high biotechnological potential of VmutPLL.

Structural Studies of a Thermophilic Esterase from a New Planctomycetes Species, Thermogutta Terrifontis.

Thermogutta terrifontis esterase (TtEst), a carboxyl esterase identified in the novel thermophilic bacterium T. terrifontis from the phylum Planctomycetes, has been cloned and over‐expressed in Escherichia coli. The enzyme has been characterized biochemically and shown to have activity towards small p‐nitrophenyl (pNP) carboxylic esters, with optimal activity for pNP‐propionate. The enzyme retained 95% activity after incubation for 1 h at 80 °C. The crystal structures of the native TtEst and its complexes with the substrate analogue d‐malate and the product acetate have been determined to high resolution. The bound ligands have allowed the identification of the carboxyl and alcohol binding pockets in the enzyme active site. Comparison of TtEst with structurally related enzymes provides insight into how differences in their catalytic activity can be rationalized based upon the properties of the amino acid residues in their active site pockets. The mutant enzymes L37A and L251A have been constructed to extend the substrate range of TtEst towards the larger butyrate and valerate pNP‐esters. These mutant enzymes have also shown a significant increase in activity towards acetate and propionate pNP esters. A crystal structure of the L37A mutant was determined with the butyrate product bound in the carboxyl pocket of the active site. The mutant structure shows an expansion of the pocket that binds the substrate carboxyl group, which is consistent with the observed increase in activity towards pNP‐butyrate.

Crystallization and preliminary X-ray diffraction analysis of ω-amino acid:pyruvate transaminase from Chromobacterium violaceum

An ω-amino acid:pyruvate transaminase from C. violaceum has been purified and crystallized in two crystal forms. The structure has been solved using molecular replacement.