Cloning and characterization of the gene encoding pyrroloquinoline quinone-dependent poly(vinyl alcohol) dehydrogenase of Pseudomonas sp. strain VM15C

Biosci Biotechnol Biochem. 1996 Jul;60(7):1056-62. doi: 10.1271/bbb.60.1056.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcohol Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Base Sequence
  • Cloning, Molecular
  • Culture Media
  • DNA, Bacterial / analysis
  • DNA, Bacterial / chemistry
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Escherichia coli / metabolism
  • Gene Library
  • Genes, Bacterial / genetics
  • Molecular Sequence Data
  • Plasmids
  • Pseudomonas / enzymology*
  • Pseudomonas / genetics*
  • Sequence Deletion

Substances

  • Culture Media
  • DNA, Bacterial
  • Alcohol Oxidoreductases
  • polyvinyl-alcohol dehydrogenase (acceptor)

Associated data

  • GENBANK/D50670