1bwk
OLD YELLOW ENZYME (OYE1) MUTANT H191N
Structural highlights
FunctionOYE1_SACPS Oxidizes beta-NADH, beta-NADPH, and alpha-NADPH. 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 PubMedOld Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme. On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194.,Brown BJ, Deng Z, Karplus PA, Massey V J Biol Chem. 1998 Dec 4;273(49):32753-62. PMID:9830019[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences | ||||||||||||||||||||
