2r14

Structure of morphinone reductase in complex with tetrahydroNAD

spinqualitylabelsall models 2r14, resolution 1.40Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 2r14 is a 1 chain structure with sequence from Pseudomonas putida. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: FMN, TXD Related: 1gwj Gene: morB (Pseudomonas putida) Resources: FirstGlance, OCA, RCSB, PDBsum Evolutionary Conservation Check, as determined by ConSurfDB. You may read the explanation of the method and the full data available from ConSurf. Publication Abstract from PubMed We have identified multiple reactive configurations (MRCs) of an enzyme-coenzyme complex that have measurably different kinetic properties. In the complex formed between morphinone reductase (MR) and the NADH analogue 1,4,5,6-tetrahydro-NADH (NADH4) the nicotinamide moiety is restrained close to the FMN isoalloxazine ring by hydrogen bonds from Asn-189 and His-186 as determined from the X-ray crystal structure. Molecular dynamic simulations indicate that removal of one of these hydrogen bonds in the N189A MR mutant allows the nicotinamide moiety to occupy a region of configurational space not accessible in wild-type enzyme. Using stopped-flow spectroscopy, we show that reduction of the FMN cofactor by NADH in N189A MR is multiphasic, identifying at least four different reactive configurations of the MR-NADH complex. This contrasts with wild-type MR in which hydride transfer occurs by environmentally coupled tunneling in a single kinetic phase [Pudney et al. J. Am. Chem. Soc. 2006, 128, 14053-14058]. Values for primary and alpha-secondary kinetic isotope effects, and their temperature dependence, for three of the kinetic phases in the N189A MR are consistent with hydride transfer by tunneling. Our analysis enables derivation of mechanistic information concerning different reactive configurations of the same enzyme-coenzyme complex using ensemble stopped-flow methods. Implications for the interpretation from kinetic data of tunneling mechanisms in enzymes are discussed. Mutagenesis of morphinone reductase induces multiple reactive configurations and identifies potential ambiguity in kinetic analysis of enzyme tunneling mechanisms.,Pudney CR, Hay S, Pang J, Costello C, Leys D, Sutcliffe MJ, Scrutton NS J Am Chem Soc. 2007 Nov 14;129(45):13949-56. Epub 2007 Oct 16. PMID:17939663 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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