Title of article :
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 Original Research Article
Author/Authors :
Stefan Krahulec، نويسنده , , Guilliano C. Armao، نويسنده , , Hansj?rg Weber، نويسنده , , Mario Klimacek، نويسنده , , Bernd Nidetzky، نويسنده ,
Issue Information :
دوهفته نامه با شماره پیاپی سال 2008
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 ≈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 °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 °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.
Keywords :
Biocatalysis , d-Mannitol 1-phosphate , Aspergillus fumigatus , Stereoselective synthesis , Mannitol-1-phosphate 5-dehydrogenase
Journal title :
Carbohydrate Research
Journal title :
Carbohydrate Research