Promiscuous (+)-γ-lactamase activity of an amidase from nitrile hydratase pathway for efficient synthesis of carbocyclic nucleosides intermediate

Based on bioinformatics analysis, the promiscuous (+)-γ-lactamase activity of an amidase was identified in Rhodococcus erythropolis PR4 and found to be involved in the nitrile hydratase pathway. The amidase is highly enantioselective and can be used in the kinetic resolution of the Vince lactam. The known structure provides a rare insight into the catalytic mechanism of (+)-γ-lactamase with absolute chiral selectivity. This lactamase was cloned, purified, biochemically characterized, and demonstrated to be an ideal catalyst for the preparation of carbocyclic nucleosides of pharmaceutical interest. The chiral selectivity of this enzyme was investigated by molecular docking and site-specific mutagenesis, which provides a foundation for further engineering of these versatile biocatalysts.     

融合双亲短肽提高腈水合酶的热稳定性研究

腈水合酶(Nitirle hydratase, NHase)催化腈类物质转化为酰胺类物质,目前用于工业生产丙烯酰胺。但在催化过程中释放的热量易导致酶分子失活。研究通过蛋白质融合技术对腈水合酶进行分子改造,提高热稳定性。将2种双亲自组装肽（self-assembling peptides, SAPs）EAK16和ELK16分别融合至恶臭假单胞菌Pseudomonas putida NRRL-18668来源NHase非催化亚基β的N末端,构建出2种融合型NHase：EAK16-NHase和ELK16-NHase。经过表达、纯化后测定酶活力,发现EAK16-NHase和ELK16 NHase的酶活力分别为(426±14) U/mg和(372±12) U/mg,保留野生型酶活力的97%和85%。在50 ℃条件下孵育0~60 min,每5 min取样后测定残存酶活力,EAK16-NHase和ELK16-NHase酶活力半衰期（T₅₀）分别为35 min和40 min,野生型NHase为20 min。说明融合EAK16和ELK16均能提高NHase的热稳定性。研究表明融合SAPs能在不显著影响酶活力的条件下提高酶的热稳定性。     

Reactions of the HCN-tetramer with aldehydes

The HCN-tetramer, a 'classic' of the prebiotic chemistry of HCN, is shown to undergo a remarkable reaction with acetaldehyde in slightly basic or neutral aqueous solution at room temperature. The reaction consists in an aldolization-type C,C-bond formation, accompanied by a (presumably aldehyde-catalyzed) hydration of one of the two nitrile groups and the formation of two cyclic aminal-type groupings, each of the latter incorporating an additional molecule of the aldehyde. Should this so far unexplored type of chemistry of the HCN-tetramer prove to have some generality, the finding might add a new dimension to the potential etiological relevance of this HCN-oligomer.     

Using a nitrilase for the surface modification of acrylic fibres

The surface of an acrylic fibre was modified with a commercial nitrilase (EC 3.5.5.1). The effect of fibre solvents and polyols on nitrilase catalysis efficiency and stability was investigated. The nitrilase action on the acrylic fabric was improved by the combined addition of 1 M sorbitol and 4% N, N-dimethylacetamide. The colour levels for samples treated with nitrilase increased 156% comparing to the control samples. When the additives were introduced in the treatment media, the colour levels increased 199%. The enzymatic conversion of nitrile groups into the corresponding carboxylic groups, on the fibre surface, was followed by the release of ammonia and polyacrylic acid. A surface erosion phenomenon took place and determined the "oscillatory" behaviour of the amount of dye uptake with time of treatment. These results showed that the outcome of the application of the nitrilase for the acrylic treatment is intimately dependent on reaction media parameters, such as time, enzyme activity and formulation.     

Z-phenylacetaldoxime degradation by a novel aldoxime dehydratase from Bacillus sp. strain OxB-1

Z-phenylacetaldoxime (Z-PAOx) degrading bacterium, identified as Bacillus sp. strain OxB-1, was isolated from soil after 2 months acclimation. The enzyme involved in the degradation of Z-PAOx was induced by the aldoxime and required FMN for its activity. The enzyme was partially purified from the cell-free extract of the strain and shown to catalyze the stoichiometric dehydration reaction of Z-PAOx to form phenylacetonitrile (PAN). Activities of nitrilase and amidase acting on PAN and phenylacetamide (PAAm), respectively, to form phenylacetate (PAA) were found in the strain grown on Z-PAOx. This is the first report of aldoxime dehydratase co-existing with nitrile degrading enzymes in bacteria.     

Chemo- and enantioselective hydrolysis of nitriles and acid amides, respectively, with resting cells of Rhodococcus sp. C3II and Rhodococcus erythropolis MP50

The two new bacterial strains, Rhodococcus sp. C3II and Rhodococcus erythropolis MP50, which have been especially selected for the enantioselective hydrolysis of pharmaceutically interesting 2-arylpropionitriles like naproxen nitrile, have been applied for the hydrolysis of various aliphatic and aromatic nitriles and acid amides. From the enantioselective hydrolysis of racemic ibuprofen amide 4, 2-phenylbutyronitrile 5a as well as the profen-related atrolactamide 8 we deduce the decisive role of both an alkyl and aryl substituent in the -position to the nitrile or amide function for high enantioselectivity of the hydrolysis. Strain C3II and MP50 differ in the activity of their nitrile hydratase–amidase enzyme systems. This is of interest for the regioselective hydrolysis of the dinitriles 10a–13a to diacids 10f–13f. While strain C3II is suitable to preferentially produce mononitrile monoamide derivatives, strain MP50 can be used especially to form mononitrile monoacid and monoamide monoacid derivatives.     

Characterization of an inducible nitrilase from a thermophilic bacillus

Nitrilase activity was induced in the thermophilic bacterium Bacillus pallidus strain Dac521 by growth on benzonitrile-supplemented minimal medium. The enzyme had a subunit relative molecular mass of 41 kDa but was purified as a complex with a putative GroEL protein (total M _r, 600 kDa). The enzyme catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitriles with widely varying k _cat/K _M values, primarily the result of differences in substrate affinity. Of the nitriles tested, 4-cyanopyridine was hydrolyzed at the fastest rate. Substitution of benzonitrile at the meta or para position either had no effect on catalytic rate or enhanced k _cat, while ortho-substitution was strongly inhibitory, probably because of steric hindrance. The effect of catalytic inhibitors was consistent with the presence of active site thiol residues although activity was little affected by putative thiol reagents such as iodoacetate, iodoacetamide, and N-methylmaleimide. Enzymatic activity was constant between pH 6 and 9 with an optimum at pH 7.6. The optimal temperature for activity was 65°C with rapid activity loss at higher temperatures. The purified nitrilase-GroEL complex had the following half-lives of activity: 8.4 h at 50°C, 2.5 h at 60°C, 13 min at 70°C, and less than 3 min at 80°C. Received: March 1, 1999 / Accepted: August 3, 1999     

Synthesis of novel pyrrolo[3,4-d]pyrazole-dicarboxylic acids and evaluation of their interaction with glutamate receptors

Chiral pyrazoline amino acids (3aR,4S,6aR)-1a and (3aR,4S,6aR)-1b, and (3aS,6S,6aS)-2a and (3aS,6S,6aS)-2b, which are conformationally constrained analogues of glutamic and homoglutamic acid, respectively, were prepared via a strategy based on the 1,3-dipolar cycloaddition of a nitrile imine to methyl N-Boc-3,4-didehydro-(S)-prolinate. The new 'amino acids' were tested for activity at ionotropic glutamate receptors. Solely the derivative (3aR,4S,6aR)-1a, which is structurally related to the previously described 4,5-dihydroisoxazole analogue (S)-CIP-A, turned out to be a potent and selective agonist for the AMPA receptors. The biological activity is due to the interaction with the orthosteric glutamate binding site.     

Discovery of selective and nonpeptidic cathepsin S inhibitors

Nonpeptidic, selective, and potent cathepsin S inhibitors were derived from an in-house pyrrolopyrimidine cathepsin K inhibitor by modification of the P2 and P3 moieties. The pyrrolopyrimidine-based inhibitors show nanomolar inhibition of cathepsin S with over 100-fold selectivity against other cysteine proteases, including cathepsin K and L. Some of the inhibitors showed cellular activities in mouse splenocytes as well as oral bioavailabilities in rats.