The mannitol dehydrogenase gene (mdh) from Leuconostoc mesenteroides is distinct from other known bacterial mdh genes.
The N-terminal amino acid sequences of the intact protein and three tryptic peptides from a 41 kDa protein purified from a commercial mannitol dehydrogenase (MDH) enzyme preparation of Leuconostoc mesenteroides ATCC-9135 were determined. Oligonucleotides deduced from these peptide sequences were used to isolate the putative mdh gene from L. mesenteroides using the Vectorette system. Nucleotide sequence analysis revealed an open reading frame (ORF1) of 1,014 bp encoding a putative MDH protein of 338 amino acids, and another open reading frame (ORF2) encoding an unknown protein of 245 amino acids. In Northern blots, a transcript of approximately 2.2-kb was detected with an mdh-specific probe. Mapping of the 5'-end of the 2.2-kb transcript indicated that mdh was the first gene of the operon. After fusion of six histidine codons to the 3'-end of the mdh gene and expression in Escherichia coli M15, active MDH was isolated using HisTrap purification. The overexpressed enzyme showed high specificity for mannitol and fructose. In dot blot hybridisation, the L. mesenteroides mdh-specific probe bound strongly to chromosomal DNA of Leuconostoc pseudomesenteroides and weakly to DNA of some heterofermentative Lactobacillus strains, whereas no hybridisation signals were obtained with DNA derived from strains carrying characterised mdh genes. Furthermore, the amino acid sequence similarity between L. mesenteroides MDH and other known MDHs was very low, suggesting that MDHs from heterofermentative lactic acid bacteria form a structurally and functionally separate enzyme group. Interestingly, L. mesenteroides MDH shared significant sequence similarity with the medium-chain dehydrogenase/reductase protein family.
Aarnikunnas
J
J
Department of Basic Veterinary Sciences, Division of Microbiology, PO Box 57, 00014 University of Helsinki, Finland.
Rönnholm
K
K
Palva
A
A
D000595:Amino Acid Sequence; D015152:Blotting, Northern; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007934:Leuconostoc / Q000201:enzymology* / Q000235:genetics*; D008354:Mannitol Dehydrogenases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D008354:Mannitol Dehydrogenases