Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp. strain VM15C.
A gene library of poly(vinyl alcohol) (PVA)-degrading Pseudomonas sp. strain VM15C was constructed in Escherichia coli with the vector pUC18. Screening of this library with a chromogenic PVA dehydrogenase assay resulted in the isolation of a clone that carries the gene (pdh) for the PVA dehydrogenase, and the entire nucleotide sequence of its structural gene was determined. The gene encodes a protein of 639 amino acid residues (68,045 Da) and in the deduced amino acid sequence, some putative functional sites, a signal sequence, a heme c-binding site, and a PQQ-binding site, were detected. The amino acid sequence showed low similarity to other types of quinoprotein dehydrogenases. PVA dehydrogenase expressed in E. coli clones required PQQ. Ca2+, and Mg2+ stimulated the activity. PVA-dependent heme c reduction occurred with exogenous PQQ in cell extracts of the E. coli clone. The PVA dehydrogenase in the E. coli clone was localized in the cytoplasm.
Shimao
M
M
Department of Biotechnology, Tottori University, Japan.
Tamogami
T
T
Nishi
K
K
Harayama
S
S
D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics*; D017384:Sequence Deletion
D003470:Culture Media; D004269:DNA, Bacterial; D000429:Alcohol Oxidoreductases; C048312:polyvinyl-alcohol dehydrogenase (acceptor)