A Single Phe54Tyr Substitution Improves the Catalytic Activity and Thermostability of Trigonopsis Variabilis D-amino Acid Oxidase

N Biotechnol. 2010 Feb 28;27(1):78-84. doi: 10.1016/j.nbt.2009.11.002. Epub 2009 Nov 10.

Abstract

The industrial importance of Trigonopsis variabilisd-amino acid oxidase (TvDAAO) is represented by its biocatalytic oxidative deamination of cephalosporin C (CPC) to yield glutaryl-7-aminocephalosporanic acid (GL-7-ACA). The process has been incorporated into a two-step bioconversion to produce 7-aminocephalosporanic acid, the crucial synthetic nucleus for several semi-synthetic cephalosporin antibiotics. A homology model of TvDAAO indicated that residue F54 is in a close proximity to the in silico docked CPC. Substitution of this F54 to Tyr (F54Y) resulted in 6-fold improvement in k(cat,app) and approximately 2.5-fold increase in K(i) of GL-7-ACA. Heat treatment (55 degrees C, 60 min) did not decrease the activity of F54Y. It is suggested that the Tyr substitution might initiate hydrogen bond formation with the amino group of CPC and facilitate deamination. Faster substrate turnover, reduced GL-7-ACA inhibition and improved thermostability of the F54Y substitution render it a useful candidate in industrial production of semi-synthetic cephems.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution*
  • Animals
  • Catalysis
  • Cephalosporins / chemical synthesis
  • Cephalosporins / chemistry
  • Cephalosporins / metabolism
  • D-Amino-Acid Oxidase* / chemistry
  • D-Amino-Acid Oxidase* / genetics
  • D-Amino-Acid Oxidase* / metabolism
  • Enzyme Stability*
  • Fungal Proteins* / genetics
  • Fungal Proteins* / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Phenylalanine / chemistry
  • Phenylalanine / genetics
  • Protein Structure, Tertiary*
  • Saccharomycetales / enzymology*
  • Sequence Alignment
  • Temperature
  • Tyrosine / chemistry
  • Tyrosine / genetics

Substances

  • Cephalosporins
  • Fungal Proteins
  • glutaryl-7-aminocephalosporanic acid
  • cephalosporin C
  • Tyrosine
  • Phenylalanine
  • 7-aminocephalosporanic acid
  • D-Amino-Acid Oxidase