Characterization of recombinant Aspergillus fumigatus mannitol-1-phosphate 5-dehydrogenase and its application for the stereoselective synthesis of protio and deuterio forms of D-mannitol 1-phosphate

Carbohydr Res. 2008 Jul 7;343(9):1414-23. doi: 10.1016/j.carres.2008.04.011. Epub 2008 Apr 10.

Abstract

A putative long-chain mannitol-1-phosphate 5-dehydrogenase from Aspergillus fumigatus (AfM1PDH) was overexpressed in Escherichia coli to a level of about 50% of total intracellular protein. The purified recombinant protein was a approximately 40-kDa monomer in solution and displayed the predicted enzymatic function, catalyzing NAD(H)-dependent interconversion of d-mannitol 1-phosphate and d-fructose 6-phosphate with a specific reductase activity of 170 U/mg at pH 7.1 and 25 degrees C. NADP(H) showed a marginal activity. Hydrogen transfer from formate to d-fructose 6-phosphate, mediated by NAD(H) and catalyzed by a coupled enzyme system of purified Candida boidinii formate dehydrogenase and AfM1PDH, was used for the preparative synthesis of d-mannitol 1-phosphate or, by applying an analogous procedure using deuterio formate, the 5-[2H] derivative thereof. Following the precipitation of d-mannitol 1-phosphate as barium salt, pure product (>95% by HPLC and NMR) was obtained in isolated yields of about 90%, based on 200 mM of d-fructose 6-phosphate employed in the reaction. In situ proton NMR studies of enzymatic oxidation of d-5-[2H]-mannitol 1-phosphate demonstrated that AfM1PDH was stereospecific for transferring the deuterium to NAD+, producing (4S)-[2H]-NADH. Comparison of maximum initial rates for NAD+-dependent oxidation of protio and deuterio forms of D-mannitol 1-phosphate at pH 7.1 and 25 degrees C revealed a primary kinetic isotope effect of 2.9+/-0.2, suggesting that the hydride transfer was strongly rate-determining for the overall enzymatic reaction under these conditions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus fumigatus / enzymology*
  • Chromatography, High Pressure Liquid
  • Deuterium / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification
  • Fungal Proteins / metabolism*
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Mannitol Phosphates / chemistry
  • Mannitol Phosphates / metabolism*
  • Molecular Structure
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism*
  • Stereoisomerism
  • Sugar Alcohol Dehydrogenases / genetics
  • Sugar Alcohol Dehydrogenases / isolation & purification
  • Sugar Alcohol Dehydrogenases / metabolism*

Substances

  • Fungal Proteins
  • Mannitol Phosphates
  • Recombinant Proteins
  • mannitol-1-phosphate
  • Deuterium
  • Sugar Alcohol Dehydrogenases
  • mannitol-1-phosphate dehydrogenase