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Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant.
J Mol Biol. 1993 May 20; 231(2):191-5.JM

Abstract

NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme.

Authors+Show Affiliations

Institut für Organische Chemie und Biochemie der Universität, Freiburg, Germany.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

8510142

Citation

Mittl, P R., et al. "Structural Differences Between Wild-type NADP-dependent Glutathione Reductase From Escherichia Coli and a Redesigned NAD-dependent Mutant." Journal of Molecular Biology, vol. 231, no. 2, 1993, pp. 191-5.
Mittl PR, Berry A, Scrutton NS, et al. Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant. J Mol Biol. 1993;231(2):191-5.
Mittl, P. R., Berry, A., Scrutton, N. S., Perham, R. N., & Schulz, G. E. (1993). Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant. Journal of Molecular Biology, 231(2), 191-5.
Mittl PR, et al. Structural Differences Between Wild-type NADP-dependent Glutathione Reductase From Escherichia Coli and a Redesigned NAD-dependent Mutant. J Mol Biol. 1993 May 20;231(2):191-5. PubMed PMID: 8510142.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Structural differences between wild-type NADP-dependent glutathione reductase from Escherichia coli and a redesigned NAD-dependent mutant. AU - Mittl,P R, AU - Berry,A, AU - Scrutton,N S, AU - Perham,R N, AU - Schulz,G E, PY - 1993/5/20/pubmed PY - 1993/5/20/medline PY - 1993/5/20/entrez SP - 191 EP - 5 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 231 IS - 2 N2 - NAD and NADP are ubiquitous coenzymes in biological redox reactions. They have distinct metabolic functions, yet they differ only by an additional phosphate group esterified at the 2'-hydroxyl group of the AMP moiety of NADP. The natural specificity of Escherichia coli glutathione reductase for NADP has previously been converted into a marked preference for NAD by introducing seven point mutations into the beta alpha beta-fold of the NADP-binding domain of the protein based on the known structure of the human enzyme. Among them was the replacement of Ala179 by glycine (A179G) in the alpha-helix of the fold, a change suggested by a difference in a sequence fingerprint previously found in the dinucleotide-binding domains of a number of dehydrogenases. Although this position is at a distance of 10 A from the bound 2'-phosphate group of NADP in glutathione reductase, the A179G mutation was found to be synergistic and beneficial. We have now carried out X-ray crystallographic analyses of the NAD-dependent mutant without and with bound NADH. A comparison of the structures of the mutant and wild-type enzymes reveals a flip of the peptide bond between Gly174 and Ala175 such that the side-chain of another introduced amino acid, Glu197, is fixed and can participate in binding the adenine ribose of NAD, thereby contributing to the ability of the mutated enzyme to exert its selectivity for the "wrong" coenzyme. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/8510142/Structural_differences_between_wild_type_NADP_dependent_glutathione_reductase_from_Escherichia_coli_and_a_redesigned_NAD_dependent_mutant_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0022-2836(83)71274-X DB - PRIME DP - Unbound Medicine ER -