Structure of chromosomal DNA coding for Pseudomonas putida S-1 salicylate hydroxylase

A gene coding for the salicylate hydroxylase has been isolated from chromosomal DNA of Pseudomonas putida S-1 and sequenced. The DNA fragment contained an open reading frame of 1266 bp encoding a polypeptide of 421 amino acid residues. The predicted amino acid sequence of the protein gave a good agreement with the sequences determined with the peptides isolated from the enzyme but methionine residue in the amino terminal was deleted in the N-terminal sequence of the enzyme protein. The nucleotide and amino acid sequences of the salicylate hydroxylase shared several common characteristics with those of the enzyme encoded on the plasmid DNA of P. putida PpG7; homology of nucleotide sequence is 58% and that of amino acid sequence is 56%. We could find two large conserved regions of the amino acid sequence at or near FAD- and NADH-binding regions. The FAD-binding site locates on the amino terminal and a lysine residue, functioning as an NADH-binding site (K. Suzuki, M. Mizuguchi, T. Gomi, and E. Itagaki, 1995, J. Biochem. 117,579-585), locates as Lys163.