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Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant.
Biochemistry. 1996 May 07; 35(18):5670-8.B

Abstract

The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+-dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity.

Authors+Show Affiliations

Laboratory of Molecular Biology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0580, USA. HURLEY@TOVE.NIDDK.NIH.GOVNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

8639526

Citation

Hurley, J H., et al. "Determinants of Cofactor Specificity in Isocitrate Dehydrogenase: Structure of an Engineered NADP+ --> NAD+ Specificity-reversal Mutant." Biochemistry, vol. 35, no. 18, 1996, pp. 5670-8.
Hurley JH, Chen R, Dean AM. Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant. Biochemistry. 1996;35(18):5670-8.
Hurley, J. H., Chen, R., & Dean, A. M. (1996). Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant. Biochemistry, 35(18), 5670-8.
Hurley JH, Chen R, Dean AM. Determinants of Cofactor Specificity in Isocitrate Dehydrogenase: Structure of an Engineered NADP+ --> NAD+ Specificity-reversal Mutant. Biochemistry. 1996 May 7;35(18):5670-8. PubMed PMID: 8639526.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant. AU - Hurley,J H, AU - Chen,R, AU - Dean,A M, PY - 1996/5/7/pubmed PY - 1996/5/7/medline PY - 1996/5/7/entrez SP - 5670 EP - 8 JF - Biochemistry JO - Biochemistry VL - 35 IS - 18 N2 - The 7-fold mutation Cys201Met/Cys332Tyr/Lys344Asp/Tyr345Ile/Val35 1Ala/Tyr391Lys/Arg395Ser converts the cofactor specificity of Escherichia coli isocitrate dehydrogenase from a 7000-fold preference for NADP+ to a 200-fold preference for NAD+, with overall activity comparable to that of wild-type NAD+-dependent isocitrate dehydrogenases. The structure of the NAD+-dependent mutant has been determined and refined to a working R-factor of 0.186 at 1.9 A resolution. The structure shows that NADP+ affinity is destroyed by removing favorable interactions between the 2'-phosphate and Tyr345, Tyr391, and Arg395 and by adding a repulsive interaction with Asp344. NAD+ affinity is enhanced by adding hydrogen bonds between Asp344 and the free 2'-hydroxyl. The favorable Asp344-2'-OH interaction requires a change in the pucker of the ribose to C2' endo and a shift in the adenine ring. The ring shift is made possible by a series of changes in steric packing interactions. The linchpin for repacking in the adenosine binding site is residue 351. The side chain of this "second layer" residue dictates packing of the surrounding "first layer" residues which interact with the 2' moiety and, in turn, directly determine specificity. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/8639526/Determinants_of_cofactor_specificity_in_isocitrate_dehydrogenase:_structure_of_an_engineered_NADP+___>_NAD+_specificity_reversal_mutant_ L2 - https://dx.doi.org/10.1021/bi953001q DB - PRIME DP - Unbound Medicine ER -