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 cloned 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 42,848. Sequencing of the first 20 N-terminal amino acid residues and of an internal part of the enzyme purified from P. putida F61 established the identity and the start codon of fdm. Comparison of the amino acid 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 second 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-like sequence upstream from the gene. The enzyme expressed in E. coli was purified to homogeneity. The molecular weight and the sequence of the 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. coli catalyzed the dismutation of formaldehyde to form methanol and formic acid at the ratio of 1:1 in the absence of the exogenous electron acceptor, NAD(H).
Yanase
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H
Department of Biotechnology, Faculty of Engineering, Tottori University, Japan.
Noda
H
H
Aoki
K
K
Kita
K
K
Kato
N
N
D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics; D017931:DNA Primers / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D015698:Genomic Library; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism
D017931:DNA Primers; D009243:NAD; D004247:DNA; D000429:Alcohol Oxidoreductases; C048530:formaldehyde dismutase; D000426:Alcohol Dehydrogenase; D015032:Zinc