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

Abstract

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 α‐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 Oγ through a hydrogen bond relay network involving the α‐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.