THE 2.5Å CRYSTAL STRUCTURE OF PENICILLIN G ACYLASE FROM A MUTANT FORM OF P. RETTGERI. Michael A. McDonough¹, Herbert E. Klei², Gayle K. Schulte³, and Judith A. Kelly¹, ¹Department of Molecular and Cell Biology, and Institute of Materials Science, University of Connecticut, Storrs, CT 06269-3125, ²Bristol-Myers Squibb, Princeton, NJ 08543-4000, ³Pfizer Central Research, Groton, CT 06340

Penicillin G acylase (EC 3.5.1.11) catalyzes the hydrolysis of the inexpensive antibiotic penicillin G into phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA). The 6-APA is used commercially in the synthesis of new penicillins with broader inhibitory profiles. A mutant form of the acylase from P. rettgeri with an altered substrate profile was crystallized in space group P6₁22 (a=b=140.5Å, c=200.2Å) from 50mM potassium phosphate, pH 7.5, 45 percent ammonium sulfate and 10 to 15 percent v/v glycerol (Klei, H. E. et al., 1995, Protein Science 4, 433-441). Native and heavy atom data have been collected to 2.5Å. The native data are 96 percent complete, and the Rsym is 11 percent on I. Data were collected at 100K. The structure has been solved by molecular replacement using AMoRe with the E. coli penicillin acylase as a model(Duggleby, H. J. et al., 1995, Nature 373, 264-268). The P. rettgeri enzyme is a heterodimer with a 23.7kDa alpha subunit, and a 62.2 kDa beta subunit with the reactive serine residue being the first residue of the beta chain. All but ten residues at the carboxyl terminus of the alpha chain have been modeled, and the native structure is being refined using X-PLOR. Currently, the R factor is 0.24, and the rms deviations in bond distances and angles are 0.011Å and 1.6^o respectively. Addition of solvent molecules to the model is in progress.

This research was supported in part by Connecticut Department of Higher Education Grant 00231-17-176, a Connecticut High Technology Scholarship to HEK, and the Program in Drug Design under a Critical Technologies grant from the State of Connecticut.