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.
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.
Krahulec
Stefan
S
Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.
Armao
Guilliano C
GC
Weber
Hansjörg
H
Klimacek
Mario
M
Nidetzky
Bernd
B
D001232:Aspergillus fumigatus / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003903:Deuterium / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008355:Mannitol Phosphates / Q000737:chemistry / Q000378:metabolism*; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D013237:Stereoisomerism; D013401:Sugar Alcohol Dehydrogenases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D005656:Fungal Proteins; D008355:Mannitol Phosphates; D011994:Recombinant Proteins; C030070:mannitol-1-phosphate; D003903:Deuterium; D013401:Sugar Alcohol Dehydrogenases; C021544:mannitol-1-phosphate dehydrogenase