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Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis.
J Biol Chem. 1993 Mar 05; 268(7):4656-60.JB

Abstract

On the basis of the crystal structure of the NAD-dependent cytoplasmic malate dehydrogenase (MDH) and its alignment with NADP-dependent counterparts, the loop region between beta-strand B and alpha-helix C in the dinucleotide-binding fold was predicted as a principal determinant for the coenzyme specificity. Two mutants, EX7 and EX3, of NAD-dependent MDH from Thermus flavus were constructed. In the EX7 mutant, the seven loop amino acids in positions 41-47, Glu-Ile-Pro-Gln-Ala-Met-Lys, were replaced by the corresponding loop residues in the NADP-dependent MDH from chloroplasts, Gly-Ser-Glu-Arg-Ser-Phe-Gln. In the EX3 mutant, Glu-41, Ile-42, and Ala-45 were substituted with the corresponding 3 amino acids in the NADP-dependent chloroplast MDH. In both mutations the coenzyme specificity was altered from NAD to NADP. Especially, the EX7 mutation resulted in a more than 1000-fold improvement in overall catalytic efficiency with NADPH and a 600-fold decrease in the efficiency with NADH as cofactors. Consequently, EX7 mutant was 132 times more efficient with NADPH than NADH without a large decrease in turnover number.

Authors+Show Affiliations

Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.No affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8444839

Citation

Nishiyama, M, et al. "Alteration of Coenzyme Specificity of Malate Dehydrogenase From Thermus Flavus By Site-directed Mutagenesis." The Journal of Biological Chemistry, vol. 268, no. 7, 1993, pp. 4656-60.
Nishiyama M, Birktoft JJ, Beppu T. Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis. J Biol Chem. 1993;268(7):4656-60.
Nishiyama, M., Birktoft, J. J., & Beppu, T. (1993). Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis. The Journal of Biological Chemistry, 268(7), 4656-60.
Nishiyama M, Birktoft JJ, Beppu T. Alteration of Coenzyme Specificity of Malate Dehydrogenase From Thermus Flavus By Site-directed Mutagenesis. J Biol Chem. 1993 Mar 5;268(7):4656-60. PubMed PMID: 8444839.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Alteration of coenzyme specificity of malate dehydrogenase from Thermus flavus by site-directed mutagenesis. AU - Nishiyama,M, AU - Birktoft,J J, AU - Beppu,T, PY - 1993/3/5/pubmed PY - 1993/3/5/medline PY - 1993/3/5/entrez SP - 4656 EP - 60 JF - The Journal of biological chemistry JO - J. Biol. Chem. VL - 268 IS - 7 N2 - On the basis of the crystal structure of the NAD-dependent cytoplasmic malate dehydrogenase (MDH) and its alignment with NADP-dependent counterparts, the loop region between beta-strand B and alpha-helix C in the dinucleotide-binding fold was predicted as a principal determinant for the coenzyme specificity. Two mutants, EX7 and EX3, of NAD-dependent MDH from Thermus flavus were constructed. In the EX7 mutant, the seven loop amino acids in positions 41-47, Glu-Ile-Pro-Gln-Ala-Met-Lys, were replaced by the corresponding loop residues in the NADP-dependent MDH from chloroplasts, Gly-Ser-Glu-Arg-Ser-Phe-Gln. In the EX3 mutant, Glu-41, Ile-42, and Ala-45 were substituted with the corresponding 3 amino acids in the NADP-dependent chloroplast MDH. In both mutations the coenzyme specificity was altered from NAD to NADP. Especially, the EX7 mutation resulted in a more than 1000-fold improvement in overall catalytic efficiency with NADPH and a 600-fold decrease in the efficiency with NADH as cofactors. Consequently, EX7 mutant was 132 times more efficient with NADPH than NADH without a large decrease in turnover number. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/8444839/Alteration_of_coenzyme_specificity_of_malate_dehydrogenase_from_Thermus_flavus_by_site_directed_mutagenesis_ L2 - http://www.jbc.org/cgi/pmidlookup?view=long&pmid=8444839 DB - PRIME DP - Unbound Medicine ER -