Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C.
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 A resolution and with bound glycerol to 2.4 A. 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 alpha-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(gamma) through a hydrogen bond relay network involving the alpha-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.
Fritz-Wolf
Karin
K
Department of Biophysics, Max-Planck Institute for Medical Research, Jahnstr. 29, D-69120 Heidelberg, Germany. fritz@mpimf-heidelberg.mpg.de
Koller
Klaus-Peter
KP
Lange
Gudrun
G
Liesum
Alexander
A
Sauber
Klaus
K
Schreuder
Herman
H
Aretz
Werner
W
Kabsch
Wolfgang
W
D000581:Amidohydrolases / Q000737:chemistry*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D002384:Catalysis; D002511:Cephalosporins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D019281:Dimerization; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010399:Penicillin Amidase / Q000737:chemistry*; D011485:Protein Binding
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; C025163:cephalosporin C; C030735:7-aminocephalosporanic acid; D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase