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Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

ABSTRACT: Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent purification. With regard to the presumptive active site residue Cys203, a Cys203 --> Ala mutant enzyme still retained 11.5% of the original specific activity. In sharp contrast, substitutions in certain other positions in the neighborhood of Cys203 had a far more dramatic effect on the amidase. Glutamic acid substitution of Asp191 reduced the specific activity of the mutant enzyme to 1.33% of the wild-type activity. Furthermore, Asp191 --> Asn substitution as well as Ser195 --> Ala substitution completely abolished the specific activity. It would thus appear that, among various conserved residues residing within the so-called signature sequence common to all amidases, the real active site residues are Asp191 and Ser195 rather than Cys203. Inasmuch as an amide bond (CO-NH2) in the amide substrate is not too far structurally removed from a peptide bond (CO-NH-), the signature sequences of various amidases were compared with the active site sequences of various types of proteases. It was found that aspartic acid and serine residues corresponding to Asp191 and Ser195 of the Rhodococcus amidase are present within the active site sequences of aspartic proteinases, thus suggesting the evolutionary relationship between the two.

SUBMITTER: Kobayashi M

PROVIDER: S-EPMC23678 | biostudies-literature | 1997 Oct

REPOSITORIES: biostudies-literature

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Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

Kobayashi M M Fujiwara Y Y Goda M M Komeda H H Shimizu S S

Proceedings of the National Academy of Sciences of the United States of America 19971001 22

Mainly based on various inhibitor studies previously performed, amidases came to be regarded as sulfhydryl enzymes. Not completely satisfied with this generally accepted interpretation, we performed a series of site-directed mutagenesis studies on one particular amidase of Rhodococcus rhodochrous J1 that was involved in its nitrile metabolism. For these experiments, the recombinant amidase was produced as the inclusion body in Escherichia coli to greatly facilitate its recovery and subsequent pu ...[more]

PMID: 9342349

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Project description:BackgroundInsight into the function of carbohydrate-active enzymes is required to understand their biological role and industrial potential. There is a need for better use of the ample genomic data in order to enable selection of the most interesting proteins for further studies. The basis for elaborating a new approach to sequence analysis is the hypothesis that when using conserved peptide patterns to determine the similarities between proteins, the exact spacing between conserved adjacent amino acids in the proteins plays a prominent functional role. Thus, the objective of developing the method of conserved unique peptide patterns (CUPP) is to construct a peptide-based grouping and validate the method to provide evidence that CUPP captures function-related features of the individual carbohydrate-active enzymes (as defined by CAZy families). This approach facilitates grouping of enzymes at a level lower than protein families and/or subfamilies. A standardized, efficient, and robust approach to functional annotation of carbohydrate-active enzymes would support improved molecular insight into enzyme-substrate interaction.ResultsA new nonalignment-based clustering and functional annotation tool was developed that uses conserved unique peptides patterns to perform automated clustering of proteins and formation of protein groups. A peptide-based model was constructed for each of these protein CUPP groups to be used to automatically annotate protein family, subfamily, and EC function of carbohydrate-active enzymes. CUPP prediction can annotate proteins (from any CAZy family) with high F-score to existing family (0.966), subfamily (0.961), and EC-function (0.843). The speed of the CUPP program was estimated and exemplified by prediction of the 504,017 nonredundant proteins of CAZy in less than four CPU hours.ConclusionIt was possible to construct an automated system for clustering proteins within families and use the resulting CUPP groups to directly build peptide-based models for genome annotation. The CUPP runtime, F-score, sensitivity, and precisions of family and subfamily annotations match or represent an improvement compared to state-of-the-art tools. The speed of the CUPP annotation is similar to the rapid DIAMOND annotation tool. CUPP facilitates automated annotation of full genome assemblies to any CAZy family.

Dataset Information

Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

Publications

Identification of active sites in amidase: evolutionary relationship between amide bond- and peptide bond-cleaving enzymes.

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