1cp9

From Proteopedia

Revision as of 12:30, 18 December 2007

CRYSTAL STRUCTURE OF PENICILLIN G ACYLASE FROM THE BRO1 MUTANT STRAIN OF PROVIDENCIA RETTGERI

Overview

Penicillin G acylase is an important enzyme in the commercial production, of semisynthetic penicillins used to combat bacterial infections. Mutant, strains of Providencia rettgeri were generated from wild-type cultures, subjected to nutritional selective pressure. One such mutant, Bro1, was, able to use 6-bromohexanamide as its sole nitrogen source. Penicillin, acylase from the Bro1 strain exhibited an altered substrate specificity, consistent with the ability of the mutant to process 6-bromohexanamide., The X-ray structure determination of this enzyme was undertaken to, understand its altered specificity and to help in the design of, site-directed mutants with desired specificities. In this paper, the, structure of the Bro1 penicillin G acylase has been solved at 2.5 A, resolution by molecular replacement. The R-factor after refinement is, 0.154 and R-free is 0.165. Of the 758 residues in the Bro1 penicillin, acylase heterodimer (alpha-subunit, 205; beta-subunit, 553), all but the, eight C-terminal residues of the alpha-subunit have been modeled based on, a partial Bro1 sequence and the complete wild-type P. rettgeri sequence. A, tightly bound calcium ion coordinated by one residue from the, alpha-subunit and five residues from the beta-subunit has been identified., This enzyme belongs to the superfamily of Ntn hydrolases and uses Ogamma, of Ser beta1 as the characteristic N-terminal nucleophile. A mutation of, the wild-type Met alpha140 to Leu in the Bro1 acylase hydrophobic, specificity pocket is evident from the electron density and is consistent, with the observed specificity change for Bro1 acylase. The electron, density for the N-terminal Gln of the alpha-subunit is best modeled by the, cyclized pyroglutamate form. Examination of aligned penicillin acylase and, cephalosporin acylase primary sequences, in conjunction with the P., rettgeri and Escherichia coli penicillin acylase crystal structures, suggests several mutations that could potentially allow penicillin acylase, to accept charged beta-lactam R-groups and to function as a cephalosporin, acylase and thus be used in the manufacture of semi-synthetic, cephalosporins.

About this Structure

1CP9 is a Protein complex structure of sequences from Providencia rettgeri with CA and SO4 as ligands. Active as Penicillin amidase, with EC number 3.5.1.11 Known structural/functional Site: . Full crystallographic information is available from OCA.

Reference

Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri., McDonough MA, Klei HE, Kelly JA, Protein Sci. 1999 Oct;8(10):1971-81. PMID:10548042

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