Tags

Type your tag names separated by a space and hit enter

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 07; 343(9):1414-23.CR

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.

Authors+Show Affiliations

Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/I, A-8010 Graz, Austria.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

18452897

Citation

Krahulec, Stefan, et al. "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." Carbohydrate Research, vol. 343, no. 9, 2008, pp. 1414-23.
Krahulec S, Armao GC, Weber H, et al. 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;343(9):1414-23.
Krahulec, S., Armao, G. C., Weber, H., Klimacek, M., & Nidetzky, B. (2008). 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. Carbohydrate Research, 343(9), 1414-23. https://doi.org/10.1016/j.carres.2008.04.011
Krahulec S, et al. 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. PubMed PMID: 18452897.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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. AU - Krahulec,Stefan, AU - Armao,Guilliano C, AU - Weber,Hansjörg, AU - Klimacek,Mario, AU - Nidetzky,Bernd, Y1 - 2008/04/10/ PY - 2008/02/01/received PY - 2008/04/02/revised PY - 2008/04/04/accepted PY - 2008/5/3/pubmed PY - 2008/10/4/medline PY - 2008/5/3/entrez SP - 1414 EP - 23 JF - Carbohydrate research JO - Carbohydr. Res. VL - 343 IS - 9 N2 - 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. SN - 0008-6215 UR - https://www.unboundmedicine.com/medline/citation/18452897/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_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0008-6215(08)00196-1 DB - PRIME DP - Unbound Medicine ER -