1iso
From Proteopedia
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ISOCITRATE DEHYDROGENASE: STRUCTURE OF AN ENGINEERED NADP+--> NAD+ SPECIFICITY-REVERSAL MUTANT
Overview
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.
About this Structure
1ISO is a Single protein structure of sequence from Escherichia coli with SO4 and AMP as ligands. Active as Isocitrate dehydrogenase (NADP(+)), with EC number 1.1.1.42 Full crystallographic information is available from OCA.
Reference
Determinants of cofactor specificity in isocitrate dehydrogenase: structure of an engineered NADP+ --> NAD+ specificity-reversal mutant., Hurley JH, Chen R, Dean AM, Biochemistry. 1996 May 7;35(18):5670-8. PMID:8639526
Page seeded by OCA on Tue Nov 20 17:38:27 2007
