Characterisation of a novel γ-lactamase enzyme used in the synthesis of an anti-HIV drug

Abstract

The human IgG antibody molecule is composed of three globular protein moieties linked through a flexible "hinge" region. Two protein moieties (Fab) determine antigen binding specificity and the third (Fc) expresses interaction sites for effector. The IgG-Fc region is a homodimer comprised of interchain disulphide bonded hinge regions, glycosylated C"2 domains and non-covalently paired cH3 domains. Glycosylation is essential for the activation of protective effector functions.We have applied differential scanning microcalorimetry to probe structural and functional relationships between homogeneous IgG-Fc glycoforms bearing progressively truncated oligosaccharides. The native IgG-Fc yields a thermogram with transition temperatures (Tm) of 7 I" and 82" for the cH2 and cH3 domains, respectively. For the truncated glycoforms the Tm of the cH2 domain is progressively lowered whilst that of the C"3 domain is not affected. This correlates with attenuation of Fyc receptor (FcyR) binding and activation.We propose that interactions of the hinge proximal primary and secondary N-acetylglucosamine sugar residues with the protein result in the generation of a population of structural conformers; amongst which are conformers specific for effector ligands'. The present studies add hrther support to this model. An enzyme responsible for the resolution of racemic y-lactam, a (+) y-lactamase was isolated from Cornomonas acidovorrins and was subsequently cloned and over-expressed in E.coli. The enzyme has been purified to homogeneity by standard chromatography techniques including hydrophobic interaction chromatography and ion exchange chromatography. The enzyme appears to be a monomer with a molecular mass of -43 kDa according to gel filtration and SDS-PAGE. The enzyme shows more than 50% sequence homology with two other enzymes, formamidase and acetamidase, and appears to have a serine residue in the active site based on inhibition studies with phenyl methyl sulfonyl fluoride and benzamidine. Crystallisation trials have been carried out with the purified enzyme and well formed crystals have been obtained with PEG as the precipitant with the addition of p-octyl glucoside.The crystals diffract to 2.8 8, and three dimensional structural determination of the enzyme is in progress. This enzyme IS currently being used to synthesise a vital building block for the synthesis of a new anti-HIV drug called Ziagen.Exeter, EX4 4QD, UK Peroxiredoxin (Prx) enzymes are preferentially expressed under conditions of stress induced by elevated levels of reactive oxygen species and reactive nitrogen species. Mammalian Prxs downregulate a range of redox-sensitive processes. Thioredoxin peroxidase B (TPx-B) is a 2-cys type II Prx and catalyses the reduction of hydrogen peroxide or alkyl hydroperoxide to water or alcohol. The 2-cys Prx mechanism involves the activity of two conserved cysteines a d uses the thioredoxin enzyme system as a source of hydrogen. The crystal structure for TPx-B has been solved to 1.7 A resolution. The refined structure is a toro...