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KMID : 0545120110210090914
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Journal of Microbiology and Biotechnology
2011 Volume.21 No. 9 p.914 ~ p.920
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Molecular Cloning and Characterization of Mannitol-1-Phosphate Dehydrogenase from Vibrio cholerae
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Prashanthi Rambhatla
Sanath Kumar
Jared T. Floyd
Manuel F. Varela
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Abstract
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Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a Km of 1.54 ± 0.1 mM and Vmax of 320.8 ± 7.81 μmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37oC, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.
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KEYWORD
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Mannitol, Vibrio cholerae, Mannitol-1-phosphate dehydrogenase
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