Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase.
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.
Brunel
F
F
Unit of Molecular Biology, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Davison
J
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D062385:Hydroxybenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000235:genetics*; D010090:Oxidoreductases, O-Demethylating / Q000235:genetics*; D011549:Pseudomonas / Q000235:genetics*; D015183:Restriction Mapping; D014641:Vanillic Acid / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D062385:Hydroxybenzoates; D010088:Oxidoreductases; D010090:Oxidoreductases, O-Demethylating; C023284:vanillate O-demethylase; D014641:Vanillic Acid