A zinc-containing mannitol-2-dehydrogenase from Leuconostoc pseudomesenteroides ATCC 12291: purification of the enzyme and cloning of the gene.
Mannitol-2-dehydrogenase (EC 1.1.1.67) of Leuconostoc pseudomesenteroides ATCC 12291 catalyzing the NADH-dependent reduction of d-fructose to d-mannitol was purified to homogeneity. Native mannitol-2-dehydrogenase has a molecular mass of 155 kDa as determined by gel filtration chromatography. In SDS-PAGE, a single band appeared corresponding to a molecular mass of 43 kDa which indicated that the enzyme was composed of four identical subunits. Enzyme activity was completely inhibited by EDTA and could be restored by zinc ions, but not by Mn(2+) or Mg(2+) which demonstrated that zinc is a cofactor. Purified mannitol-2-dehydrogenase exhibited a maximal specific activity of 400 micromol fructose reduced min(-1) x (mg protein)(-1), using NADH as electron donor. The enzyme showed a high substrate specificity for d-fructose and d-mannitol, however it accepted NADPH as a cofactor with 32% activity ( V(max)) relative to NADPH (100%). The mdh gene, encoding mannitol-2-dehydrogenase, was identified by hybridization with a degenerate gene probe complementary to the nucleotide sequence encoding the first eight N-terminal amino acids of the enzyme. The mdh gene was cloned on a 4.2-kb DNA fragment, subcloned, and expressed in Escherichia coli. Sequencing of the gene revealed an open reading frame of 1017 bp, encoding a protein of 338 amino acids with a predicted molecular mass of 36.0 kDa. Plasmid-encoded mdh was functionally expressed, with 70 U/mg of cell-free protein in E. coli. Multiple sequence alignments showed that mannitol-2-dehydrogenase was affiliated with members of the Zn(2+)-containing medium-chain alcohol/polyol dehydrogenase/reductase protein family (MDR).
Hahn
Gerald
G
Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany.
Kaup
Björn
B
Bringer-Meyer
Stephanie
S
Sahm
Hermann
H
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D007934:Leuconostoc / Q000201:enzymology*; D008354:Mannitol Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D015032:Zinc / Q000032:analysis*
D000429:Alcohol Oxidoreductases; D008354:Mannitol Dehydrogenases; C116789:mannitol 2-dehydrogenase (NADP); D015032:Zinc