1ISO

ISOCITRATE DEHYDROGENASE: STRUCTURE OF AN ENGINEERED NADP+--> NAD+ SPECIFICITY-REVERSAL MUTANT

1ISO の概要

• エントリーDOI: 10.2210/pdb1iso/pdb
• 分子名称: ISOCITRATE DEHYDROGENASE, SULFATE ION, NICOTINAMIDE-ADENINE-DINUCLEOTIDE, ... (4 entities in total)
• 機能のキーワード: nadp, phosphorylation, glyoxylate bypass, oxidoreductase
• 由来する生物種: Escherichia coli
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 46652.99

構造登録者

Hurley, J.H. (登録日: 1996-03-01, 公開日: 1996-12-07, 最終更新日: 2024-05-22)

主引用文献

Hurley, J.H.,Chen, R.,Dean, A.M.
Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant.
Biochemistry, 35:5670-5678, 1996

PubMed 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.

PubMed: 8639526
DOI: 10.1021/bi953001q

実験手法

X-RAY DIFFRACTION (1.9 Å)