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

Protein Sci. 2002 Jan;11(1):92-103. doi: 10.1110/ps.27502.

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 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.

MeSH terms

  • Amidohydrolases / chemistry*
  • Amino Acid Sequence
  • Anti-Bacterial Agents / chemistry*
  • Catalysis
  • Cephalosporins / chemistry*
  • Crystallography, X-Ray
  • Dimerization
  • Models, Chemical
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Penicillin Amidase / chemistry*
  • Protein Binding

Substances

  • Anti-Bacterial Agents
  • Cephalosporins
  • cephalosporin C
  • 7-aminocephalosporanic acid
  • Amidohydrolases
  • Penicillin Amidase
  • glutarylamidocephalosporanic acid acylase

Associated data

  • PDB/1GK0
  • PDB/1GK1