Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

Appl Microbiol Biotechnol. 2008 Feb;78(1):75-83. doi: 10.1007/s00253-007-1286-0. Epub 2007 Dec 11.

Abstract

Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA ( Cg ) (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA (Cg) was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobA(Cg). The pobA (Cg) is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Binding Sites
  • Corynebacterium glutamicum / enzymology*
  • Corynebacterium glutamicum / genetics
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Gene Order
  • Genes, Bacterial
  • Hydroxybenzoates / metabolism
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / isolation & purification*
  • Mixed Function Oxygenases / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • NAD / metabolism
  • NADP / metabolism
  • Operon
  • Parabens / metabolism*
  • Protein Binding
  • Substrate Specificity

Substances

  • 3,4-dihydroxybenzoate
  • Hydroxybenzoates
  • Parabens
  • NAD
  • NADP
  • Mixed Function Oxygenases
  • 4-hydroxybenzoic acid