Lactobacillus reuteri ATCC 53608 mdh gene cloning and recombinant mannitol dehydrogenase characterization.
A gene encoding mannitol-2-dehydrogenase (E.C. 1.1.1.138) (MDH) was cloned from Lactobacillus reuteri and expressed in Escherichia coli. The 1,008-bp gene encodes a protein consisting of 336 amino acids, with a predicted molecular mass of 35,920 Da. The deduced amino acid sequence of L. reuteri MDH (LRMDH) is 77% and 76% similar to the MDHs from Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, respectively. The purified recombinant enzyme appears as a single band of 40 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but gel filtration indicates that the native enzyme is a dimer. The optimum temperature for the recombinant enzyme is 37 degrees C, the pH optima for D-fructose reduction and D-mannitol oxidation are 5.4 and 6.2, respectively. The K(m) values for NAD (9 mM) and NADH (0.24 mM) are significantly higher than those for NADP (0.35 mM) and NADPH (0.04 mM). The K(m) values of LRMDH for D-fructose and D-mannitol are 34 mM and 54 mM, respectively. Contrary to what the enzyme sequence suggests, recombinant LRMDH contains a single catalytic zinc per subunit.
Sasaki
Yoshiharu
Y
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Laivenieks
Maris
M
Zeikus
J Gregory
JG
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics; D008354:Mannitol Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008354:Mannitol Dehydrogenases; D015032:Zinc