Cloning, nucleotide sequence and expression of a mannitol dehydrogenase gene from Pseudomonas fluorescens DSM 50106 in Escherichia coli

Biochim Biophys Acta. 1997 Mar 20;1351(1-2):157-67. doi: 10.1016/s0167-4781(96)00189-3.

Abstract

A NAD-dependent mannitol dehydrogenase (MtlD) was purified to homogeneity from P. fluorescens DSM50106 and the N-terminal amino acid sequence was determined. An oligonucleotide deduced from this peptide sequence was used as a probe to isolate the mannitol dehydrogenase gene (mtlD) from a genomic library of P. fluorescens. Nucleotide sequence analysis of a 1.8 kb NruI fragment containing the entire mtlD gene revealed an open reading frame of 1482 bp encoding a protein with a calculated molecular weight of 54.49 kDa. The enzyme shared a high similarity with a mannitol dehydrogenase from Rhodobacter sphaeroides and a putative mannitol dehydrogenase of Saccharomyces cerevisae with an overall identity in amino acid sequence of 44% and 42%, respectively, whereas the similarity to mannitol-1-phosphate dehydrogenases of Escherichia coli or Enterococcus faecalis was only about 23% of identical amino acids. By construction of inducible expression plasmids the specific activity of the mannitol dehydrogenase synthesized in E. coli was increased from 0.02 U (mg protein)(-1) to 10 U (mg protein)(-1). After fusion of six histidine codons to the 3' end of mtlD gene and expression in E. coli active mannitol dehydrogenase could be purified in a two-step procedure by affinity chromatography using a Ni2+ matrix column. The purified enzyme exhibited a specific activity of 46 U (mg protein)(-1) and was shown to be a polyol dehydrogenase with a broad substrate spectrum oxidizing efficiently mannitol, sorbitol and arabitol.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Base Sequence
  • Chromatography, Affinity
  • Escherichia coli / genetics
  • Genes, Bacterial*
  • Genomic Library
  • Histidine*
  • Mannitol Dehydrogenases / genetics*
  • Mannitol Dehydrogenases / isolation & purification
  • Molecular Sequence Data
  • NAD / metabolism
  • NADP / metabolism
  • Peptides / genetics
  • Protein Engineering
  • Pseudomonas fluorescens / enzymology
  • Pseudomonas fluorescens / genetics*
  • Recombinant Proteins / isolation & purification
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • Peptides
  • Recombinant Proteins
  • NAD
  • polyhistidine
  • Histidine
  • NADP
  • Mannitol Dehydrogenases

Associated data

  • GENBANK/AF007800