Cloning and high-level expression of the glutathione-independent formaldehyde dehydrogenase gene from Pseudomonas putida.
A DNA fragment of 485 bp was specifically amplified by PCR with primers based on the N-terminal sequence of the purified formaldehyde dehydrogenase (EC 1.2.1.46) from Pseudomonas putida and on that of a cyanogen bromide-derived peptide. With this product as a probe, a gene coding for formaldehyde dehydrogenase (fdhA) in P. putida chromosomal DNA was cloned in Escherichia coli DH5 alpha. Sequencing analysis revealed that the fdhA gene contained 1,197-bp open reading frame, encoding a protein composed of 399 amino acid residues whose calculated molecular weight was 42,082. The transformant of E. coli DH5 alpha harboring the hybrid plasmid, pFDHK3DN71, showed about 50-fold-higher formaldehyde dehydrogenase activity than P. putida. The predicted amino acid sequence contained several features characteristic of the zinc-containing medium-chain alcohol dehydrogenase (ADH) family. Most of the glycine residues strictly conserved within the family, including a Gly-Xaa-Gly-Xaa-Xaa-Gly pattern in the coenzyme binding domain, were well conserved in this enzyme. Regions around both the catalytic and the structural zinc atoms were also conserved. Analyses of structural and enzymatic characteristics indicated that P. putida FDH belongs to the medium-chain ADH family, with mixed properties of mammalian class I and III ADHs.
Ito
K
K
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Takahashi
M
M
Yoshimoto
T
T
Tsuru
D
D
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D005557:Formaldehyde / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D015032:Zinc
D017931:DNA Primers; D008667:Metalloproteins; D011994:Recombinant Proteins; D005557:Formaldehyde; D000426:Alcohol Dehydrogenase; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent; D015032:Zinc