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Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C

¹ Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany
² Aventis Pharma Deutschland GMBH, D-65926 Frankfurt am Main, Germany

Reprint requests to: Karin Fritz-Wolf, Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany; e-mail: fritz{at}mpimf-heidelberg.mpg.de; fax: 49-6221-486-437.

Glutarylamidase is an important enzyme employed in the commercial production of 7-aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7-aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two-step enzymatic process utilizing the enzymes D-amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7-aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 Å resolution and with bound glycerol to 2.4 Å. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side-chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the -amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the O through a hydrogen bond relay network involving the -amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid.

Keywords: Cephalosporin acylase; glutaryl acylase; cephalosporin C; catalytic triad; Ntn-hydrolase; X-ray structure

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