C-terminus mutations of Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase with improved activity toward penicillin analogs

FEMS Microbiol Lett. 2005 May 1;246(1):103-10. doi: 10.1016/j.femsle.2005.03.043.

Abstract

Deacetoxy/deacetylcephalosporin C synthase (acDAOC/DACS) from Acremonium chrysogenum is a bifunctional enzyme that catalyzes both the ring-expansion of penicillin N to deacetoxycephalosporin C (DAOC) and the hydroxylation of the latter to deacetylcephalosporin C (DAC). Three residues N305, R307 and R308 located in close proximity to the C-terminus of acDAOC/DACS were each mutated to leucine. The N305L and R308L mutant acDAOC/DACSs showed significant improvement in their ability to convert penicillin analogs. R308 was identified for the first time as a critical residue for DAOC/DACS activity. Kinetic analyses of purified R308L enzyme indicated its improved catalytic efficiency is due to combined improvements of K(m) and k(cat). Comparative modeling of acDAOC/DACS supports the involvement of R308 in the formation of substrate-binding pocket.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acremonium / enzymology*
  • Acremonium / genetics
  • Amino Acid Substitution
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation*
  • Oxygenases / chemistry
  • Oxygenases / genetics*
  • Oxygenases / isolation & purification
  • Oxygenases / metabolism*
  • Penicillins / metabolism*

Substances

  • Penicillins
  • Oxygenases
  • deacetoxycephalosporin C hydroxylase