Aliphatic Amidase from Rhodococcus rhodochrous M8 Is Related to the Nitrilase/Cyanide Hydratase Family

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S. I. Pertsovich¹, D. T. Guranda¹, D. A. Podchernyaev², A. S. Yanenko², and V. K. Svedas^1*

¹Faculty of Bioengineering and Bioinformatics, Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia; fax: (7-095) 939-2355; E-mail: vytas@belozersky.msu.ru

²Institute of Genetics and Selection of Industrial Microorganisms, 1-yi Dorozhnyi Proezd 1, 113545 Moscow, Russia

^* To whom correspondence should be addressed.

Received March 2, 2005; Revision received March 15, 2005

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A comparative study of amino acid sequence and physicochemical properties indicates the affiliation of an amidase from Rhodococcus rhodochrous M8 (EC 3.5.1.4) to the nitrilase/cyanide hydratase family. Cluster analysis and multiple alignments show that Cys166 is an active site nucleophile. The enzyme has been shown to be a typical aliphatic amidase, being the most active toward short-chain linear amides. Small polar molecules such as hydroxylamine and O-methyl hydroxylamine can serve as effective external nucleophiles in acyl transfer reactions. The kinetics of the industrially important amidase-catalyzed acrylamide hydrolysis has been studied over a wide range of substrate concentrations; inhibition during enzymatic hydrolysis by the substrate and product (acrylic acid) has been observed; an adequate kinetic scheme has been evaluated and the corresponding kinetic parameters have been determined.

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KEY WORDS: Rhodococcus rhodochrous amidase, sequence alignment, amidase classification, substrate specificity, acrylamide hydrolysis

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