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Structure‐based prediction of modifications in glutarylamidase to allow single‐step enzymatic production of 7‐aminocephalosporanic acid from cephalosporin C
Author(s) -
FritzWolf Karin,
Koller KlausPeter,
Lange Gudrun,
Liesum Alexander,
Sauber Klaus,
Schreuder Herman,
Aretz Werner,
Kabsch Wolfgang
Publication year - 2002
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.27502
Subject(s) - cephalosporin c , penicillin amidase , chemistry , serine , cephalosporin antibiotic , enzyme , stereochemistry , histidine , biochemistry , combinatorial chemistry , cephalosporin , immobilized enzyme , antibiotics
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.