The 1.8Å Crystal Structure of Penicillin G Acylase from the Bro1 mutant of Providencia rettgeri. Michael A. McDonough1, Herbert E. Klei2, 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
The 1.8Å resolution native crystal structure of the Bro1 mutant form of penicillin G acylase (PA) is presented along with the structure of Bro1 PA complexed with the reaction product 6-bromohexanoic acid at 2.8Å resolution.
PA is an amidase that hydrolyzes the side chain of the (-lactam antibiotic, penicillin G, into phenylacetic acid and 6-aminopenicillanic acid (6-APA). 6-APA is subsequently used in the manufacture of semisynthetic penicillins. PA is a 94kDa heterodimer enzyme with 205 residues in the ( chain and 553 residues in the ( chain. Selective pressure using 6-bromohexanamide (BrHex) as the nitrogen source was part of a master plan to select for a mutant PA that would accept a larger zwitterionic side chain. The Bro1 mutant failed to evolve further to the point when it would use 6-carboxlyhexanamide (CbHex) as its only nitrogen source.
Both the native and complex structures indicate a mutation in the specificity pocket of Met(140 to Leu. The location of this mutation is consistent with specificity of Bro1 toward the substrate BrHex. These structural studies provide an explanation as to why it would be difficult to obtain mutations via selective pressure that would allow for the acceptance of CbHex. They also provide insight toward site-directed mutations which may lead to an enzyme with CbHex amidase activity.
Data for the native Bro1were collected at the SSRL beamline 7-1 using 1.08Å radiation. The enzyme was crystallized in spacegroup P6122 with unit cell dimensions a=b=140.7Å c=200.0Å. A total of 34,161 unique reflections were collected with Rsym=3.8% on I and 87.2% completeness in the last resolution shell. The native structure was solved by molecular replacement using the PA from E.coli 1 as a search model and refinement is in progress.
1. Duggleby, H.J., et al. Nature. 373, 264. (1995).