Deacylation activity of cephalosporin acylase to cephalosporin C is improved by changing the side-chain conformations of active-site residues

Bora Oh, Myungsook Kim, Jongchul Yoon, Kyungwha Chung, Yongchul Shin, Dongsoon Lee, Youngsoo Kim

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44 Scopus citations

Abstract

Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), mainly by environmentally toxic chemical deacylation of cephalosporin C (CPC). Thus, the enzymatic conversion of CPC to 7-ACA by cephalosporin acylase (CA) would be very interesting. However, CAs use glutaryl-7-ACA (GL-7-ACA) as a primary substrate and the enzymes have low turnover rates for CPC. The active-site residues of a CA were mutagenized to various residues to increase the deacylation activity of CPC, based on the active-site conformation of the CA structure. The aim was to generate sterically favored conformation of the active-site to accommodate the D-α-aminoadipyl moiety of CPC, the side-chain moiety that corresponds to the glutaryl moiety of GL-7-ACA. A triple mutant of the CA, Q50β M/Y149αK/F177βG, showed the greatest improvement of deacylation activity to CPC up to 790% of the wild-type. Our current study is an efficient method for improving the deacylation activity to CPC by employing the structure-based repetitive saturation mutagenesis.

Original languageEnglish
Pages (from-to)19-27
Number of pages9
JournalBiochemical and Biophysical Research Communications
Volume310
Issue number1
DOIs
StatePublished - 10 Oct 2003

Keywords

  • 7-Aminocephalosporanic acid
  • Cephalosporin acylase
  • Glutaryl-7-aminocephalosporanic acid
  • Penicillin acylase
  • Protein engineering
  • Substrate specificity

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