1get
|
ANATOMY OF AN ENGINEERED NAD-BINDING SITE
OverviewOverview
The coenzyme specificity of Escherichia coli glutathione reductase was, switched from NADP to NAD by modifying the environment of the 2'-phosphate, binding site through a set of point mutations: A179G, A183G, V197E, R198M, K199F, H200D, and R204P (Scrutton NS, Berry A, Perham RN, 1990, Nature, 343:38-43). In order to analyze the structural changes involved, we have, determined 4 high-resolution crystal structures, i.e., the structures of, the wild-type enzyme (1.86 A resolution, R-factor of 16.8%), of the, wild-type enzyme ligated with NADP (2.0 A, 20.8%), of the NAD-dependent, mutant (1.74 A, 16.8%), and of the NAD-dependent mutant ligated with NAD, (2.2 A, 16.9%). A comparison of these structures reveals subtle, differences that explain details of the specificity change. In particular, a peptide rotation occurs close to the adenosine ribose, with a, concomitant change of the ribose pucker. The mutations cause a contraction, of the local chain fold. Furthermore, the engineered NAD-binding site, assumes a less rigid structure than the NADP site of the wild-type enzyme., A superposition of the ligated structures shows a displacement of NAD, versus NADP such that the electron pathway from the nicotinamide ring to, FAD is elongated, which may explain the lower catalytic efficiency of the, mutant. Because the nicotinamide is as much as 15 A from the sites of the, mutations, this observation reminds us that mutations may have important, long-range consequences that are difficult to anticipate.
About this StructureAbout this Structure
1GET is a Single protein structure of sequence from Escherichia coli with FAD and NAP as ligands. Active as Glutathione-disulfide reductase, with EC number 1.8.1.7 Full crystallographic information is available from OCA.
ReferenceReference
Anatomy of an engineered NAD-binding site., Mittl PR, Berry A, Scrutton NS, Perham RN, Schulz GE, Protein Sci. 1994 Sep;3(9):1504-14. PMID:7833810
Page seeded by OCA on Tue Nov 20 15:59:55 2007