Cloning and Sequencing of Pseudomonas Genes Encoding Vanillate Demethylase

J Bacteriol. 1988 Oct;170(10):4924-30. doi: 10.1128/jb.170.10.4924-4930.1988.

Abstract

A 2,598-base-pair (bp) SalI-HincII DNA fragment has been cloned which codes for vanillate demethylase, the enzyme responsible for the demethylation of vanillate (3-methoxy-4-hydroxybenzoate) to protocatechuate (3,4-dihydroxybenzoate). Complementation and insertional inactivation experiments have shown that this fragment carries two genes (vanA and vanB) which are predominantly cotranscribed from a promoter upstream of vanA. Nucleotide sequencing of the SalI-HincII fragment confirmed the genetic data: two open reading frames of 987 and 942 bp were present in the transcribed orientation. These had a very high G + C content in the third base of each codon, which is characteristic of Pseudomonas chromosomal genes. Expression of the genes in Escherichia coli with the T7 RNA polymerase-promoter system gave rise to two polypeptides of 36 and 33 kilodaltons which could be identified by deletion analysis as the products of vanA and vanB, respectively. A search of the protein sequence data bank indicated that the vanB gene product was related to the ferredoxin family.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Base Sequence
  • Cloning, Molecular
  • DNA, Bacterial / genetics
  • Gene Expression Regulation
  • Genes, Bacterial*
  • Hydroxybenzoates / metabolism*
  • Molecular Sequence Data
  • Oxidoreductases / genetics*
  • Oxidoreductases, O-Demethylating / genetics*
  • Pseudomonas / genetics*
  • Restriction Mapping
  • Vanillic Acid / metabolism*

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Hydroxybenzoates
  • Oxidoreductases
  • Oxidoreductases, O-Demethylating
  • vanillate O-demethylase
  • Vanillic Acid

Associated data

  • GENBANK/M22077