CLONING, SEQUENCE-ANALYSIS, AND EXPRESSION OF THE GENE ENCODING FORMALDEHYDE DISMUTASE FROM PSEUDOMONAS-PUTIDA F61

The gene (fdm) coding for formaldehyde dismutase (EC 1.2.99.4) from a genomic library of formaldehyde-tolerant Pseudomonas putida F61 was cl oned and expressed in Escherichia coli. The nucleotides of the cloned DNA were sequenced; they included a single open reading frame of 1200 base pairs, coding for a putative protein with a molecular weight of 4 2,848. Sequencing of the first 20 N-terminal amino acid residues and o f an internal part of the enzyme purified from P. putida F61 establish ed the identity and the start codon of fdm. Comparison of the amino ac id sequence predicted from fdm with that of alcohol dehydrogenase from horse liver suggested a putative pyridine-dinucleotide-binding domain in fdm, and also potential ligands for the catalytic domain and the s econd zinc atom-folding domain. fdm seemed to be expressed in E. coli under control of the promoter of fdm; there was an E. coli promoter-li ke sequence upstream from the gene. The enzyme expressed in E. coli wa s purified to homogeneity. The molecular weight and the sequence of th e first 20 N-terminal amino acid residues were identical with those of P. putida formaldehyde dismutase. Each subunit contained 1 mol of NAD (H) and 2 mol of zinc per mol of protein. The enzyme produced in E. co li catalyzed the dismutation of formaldehyde to form methanol and form ic acid at the ratio of 1:1 in the absence of the exogenous electron a cceptor, NAD(H).