Amidase

Nitrile-converting enzymes are becoming commonplace in the synthesis of pharmaceuticals and commodity chemicals. These versatile biocatalysts have potential applications in different fields including synthetic biocatalysis and bioremediation. This review attempts to describe in detail the three major classes of nitrile-converting enzymes, namely nitrilases, nitrile hydratases and amidases. Various aspects of these enzymes including their occurrence, mechanism of action, characteristics and applicability in different sectors have been elaborately elucidated. Cloning of genes related to nitrile-converting enzymes is also discussed.

Escherichia coli contains multiple peptidoglycan-specific hydrolases, but their physiological purposes are poorly understood. Several mutants lacking combinations of hydrolases grow as chains of unseparated cells, indicating that these enzymes help cleave the septum to separate daughter cells after cell division. Here, we confirm previous observations that in the absence of two or more amidases, thickened and dark bands, which we term septal peptidoglycan (SP) rings, appear at division sites in isolated sacculi. The formation of SP rings depends on active cell division, and they apparently represent a cell division structure that accumulates because septal synthesis and hydrolysis are uncoupled. Even though septal constriction was incomplete, SP rings exhibited two properties of mature cell poles: they behaved as though composed of inert peptidoglycan, and they attracted the IcsA protein. Despite not being separated by a completed peptidoglycan wall, adjacent cells in these chains were often compartmentalized by the inner membrane, indicating that cytokinesis could occur in the absence of invagination of the entire cell envelope. Finally, deletion of penicillin-binding protein 5 from amidase mutants exacerbated the formation of twisted chains, producing numerous cells having septa with abnormal placements and geometries. The results suggest that the amidases are necessary for continued peptidoglycan synthesis during cell division, that their activities help create a septum having the appropriate geometry, and that they may contribute to the development of inert peptidoglycan.

BfpA, the structural repeating protein subunit A of the bundle-forming pilus and EspB, a type-III-secreted pore-forming protein of enteropathogenic Escherichia coli (EPEC), both virulence factors central for EPEC pathogenesis, were overexpressed in E. coli DH5α and M15 laboratory strains, respectively, using the pQE-30 cloning expression system, as chimeric fusions to a NH2-terminal histidine hexapeptide (His6-tag) sequence. After isopropyl β-d-thiogalactoside induction, the expression levels achieved were 11 and 40% of total soluble protein for BfpA and EspB, respectively. The His6-tagged recombinant proteins were purified (up to 98% homogeneity) by Ni-agarose affinity chromatography and produced yields varying from 0.65 to 3.1 mg of recombinant protein per gram of wet weight cells. The immunogenicity and antigenicity of the final products were tested in rabbits and using fecal specimens obtained from children suffering from acute watery diarrhea, respectively. The recombinant products correspond to antigenically authentic protein standards, useful in future epidemiological and neonatal vaccinology studies.

Fatty acid amide hydrolase (FAAH) is a promising target for modulating endocannabinoid and fatty acid ethanolamide signaling, which may have important therapeutic potential. We recently described a new class of O-arylcarbamate inhibitors of FAAH, including the cyclohexylcarbamic acid biphenyl-3-yl ester URB524 (half-maximal inhibitory concentration, IC(50) = 63 nM), which have significant anxiolytic-like properties in rats. In the present study, by introducing a selected group of substituents at the meta and para positions of the distal phenyl ring of URB524, we have characterized structure-activity profiles for this series of compounds and shown that introduction of small polar groups in the meta position greatly improves inhibitory potency. Most potent in the series was the m-carbamoyl derivative URB597 (4i, IC(50) = 4.6 nM). Furthermore, quantitative structure-activity relationship (QSAR) analysis of an extended set of meta-substituted derivatives revealed a negative correlation ...