Molecular Cloning and Characterization of Mannitol-1-phosphate dehydrogenase from Vibrio cholerae

The polyol, D-mannitol, is abundant in nature and can be found in plants, algae, fungi, and bacteria. Consequently, many microorganisms have evolved physiological capabilities to metabolize mannitol and thereby derive energy. Mannitol is also a major storage polysaccharide in fungi and plays important roles in osmoregulation and stress tolerance [13, 24]. Many microorganisms have mannitol utilization systems, mainly of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) types. The genes participating in mannitol uptake and utilization are organized in operons in both Gram-positive and Gram–negative bacteria [1, 7, 16]. In E. coli, mannitol is simultaneously phosphorylated and taken up by PTS proteins to form mannitol 1-phsophate, which is subsequently oxidized to fructose 6-phosphate (Fru6P) by a mannitol phosphate dehydrogenase enzyme (MPD) (EC 1.1.1.17) [17, 29]. Polyol dehydrogenases are broadly classified, based on the sequence analysis, into three groups; long chain dehydrogenases with more than 450 amino acids, medium chain dehydrogenases with up to 350 amino acids, and short chain dehydrogenases with up to 250 amino acids [14, 19, 21]. MtlD has been studied in various Gram-positive and Gram–negative bacteria, and it has been observed that all belong to the medium chain dehydrogenase group [4, 8, 11, 18, 27]. A novel MPD, with broad substrate specificity, has been described from Pseudomonas fluorescence [5]. Genome sequence analysis of V. cholerae O395 has revealed that the mannitol operon genome sequence is arranged as 3 complete open reading frames (ORFs) over a stretch of 3945 bp [6]. The ORFs are identified as mtlA, encoding a mannitol enzyme IICBA component (EIIMtl), mtlD, encoding a mannitol-1-phosphate dehydrogenase, and mtlR, encoding a putative operon repressor (MtlR). Recently, we cloned this entire operon and studied the mannitol transport activity in an E. coli background [15]. In the present study, we describe the cloning, purification, and functional characterization of a mannitol-1-phosphate dehydrogenase from V. cholerae O395.