Analysis of a substrate specificity switch residue of cephalosporin acylase

Charles F. Sio, Linda G. Otten, Robbert H. Cool, Wim J. Quax

Research output: Contribution to journalArticleScientificpeer-review

19 Citations (Scopus)

Abstract

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency (k cat/Km) of mutant SY-77F375C towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of Km. The kcat of mutant SY-77 F375H towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.

Original languageEnglish
Pages (from-to)755-760
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume312
Issue number3
DOIs
Publication statusPublished - 19 Dec 2003

Keywords

  • Adipyl-7-ADCA
  • Cephalosporin acylase
  • Glutaryl acylase
  • Protein engineering
  • Pseudomonas SY-77
  • Saturation mutagenesis
  • Semi-synthetic cephalosporins
  • Site directed mutagenesis
  • Substrate specificity

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