tailieunhanh - Báo cáo khoa học: Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP+

Formate dehydrogenase from Candida boidinii(CboFDH) catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD + to NADH. CboFDH is highly specific to NAD + and virtually fails to catalyze the reaction with NADP + . Based on structural informa-tion for CboFDH, the loop region betweenb-sheet 7 and a-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coen-zyme specificity. | Structure-guided alteration of coenzyme specificity of formate dehydrogenase by saturation mutagenesis to enable efficient utilization of NADP Aggeliki Andreadeli1 Dimitris Platis1 Vladimir Tishkov2 Vladimir Popov3 and Nikolaos E. Labrou1 1 Laboratory of Enzyme Technology Department of AgriculturalBiotechnology AgriculturalUniversity of Athens Greece 2 Department of ChemicalEnzymology Faculty of Chemistry . Lomonosov Moscow State University Russia 3 Institute of Biochemistry Russian Academy of Sciences Moscow Russia Keywords coenzyme coenzyme specificity enzyme redesign formate dehydrogenase saturation mutagenesis Correspondence N. E. Labrou Laboratory of Enzyme Technology Department of Agricultural Biotechnology AgriculturalUniversity of Athens 75 Iera Odos GR 118 55 Athens Greece Fax Tel 30 210 5294308 E-mail lambrou@ Received 18 February 2008 revised 29 May 2008 accepted 2 June 2008 doi Formate dehydrogenase from Candida boidinii CboFDH catalyses the oxidation of formate anion to carbon dioxide with concomitant reduction of NAD to NADH. CboFDH is highly specific to NAD and virtually fails to catalyze the reaction with NADP . Based on structural information for CboFDH the loop region between b-sheet 7 and a-helix 10 in the dinucleotide-binding fold was predicted as a principal determinant of coenzyme specificity. Sequence alignment with other formate dehydrogenases revealed two residues Asp195 and Tyr196 that could account for the observed coenzyme specificity. Positions 195 and 196 were subjected to two rounds of site-saturation mutagenesis and screening and enabled the identification of a double mutant Asp195Gln Tyr196His. which showed a more than 2 X 107-fold improvement in overall catalytic efficiency with NADP and a more than 900-fold decrease in the efficiency with NAD as cofactors. The results demonstrate that the combined polar interactions and steric factors comprise the main structural determinants responsible .

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