1CP9

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

Summary for 1CP9

• Entry DOI: 10.2210/pdb1cp9/pdb
• Descriptor: Penicillin G amidase, SULFATE ION, CALCIUM ION, ... (5 entities in total)
• Functional Keywords: antibiotic resistance, amidohydrolase, ntn-hydrolase fold, n-terminal pyroglutamate, penicillin binding protein, calcium binding protein, hydrolase
• Biological source: Providencia rettgeri; More
• Total number of polymer chains: 2
• Total formula weight: 86357.50

Authors

McDonough, M.A.,Klei, H.E.,Kelly, J.A. (deposition date: 1999-06-12, release date: 1999-06-24, Last modification date: 2024-10-16)

Primary citation

McDonough, M.A.,Klei, H.E.,Kelly, J.A.
Crystal structure of penicillin G acylase from the Bro1 mutant strain of Providencia rettgeri.
Protein Sci., 8:1971-1981, 1999

PubMed Abstract: 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.

PubMed: 10548042

Experimental method

X-RAY DIFFRACTION (2.5 Å)