Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Characterization of Mannitol-1-Phosphate Dehydrogenase from Vibrio cholerae

  • Rambhatla, Prashanthi (Eastern New Mexico University, Department of Biology) ;
  • Kumar, Sanath (Eastern New Mexico University, Department of Biology) ;
  • Floyd, Jared T. (Eastern New Mexico University, Department of Biology) ;
  • Varela, Manuel F. (Eastern New Mexico University, Department of Biology)
  • Received : 2011.04.14
  • Accepted : 2011.06.14
  • Published : 2011.09.28
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Abstract

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 $6{\times}$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 $K_m$ of $1.54{\pm}0.1$ mM and $V_{max}$ of $320.8{\pm}7.81\;{\mu}mol$/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and $37^{\circ}C$, 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.

Keywords

References

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