On the Active Site of Old Yellow Enzyme

Abstract

Old 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 ␣,␤-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. Old Yellow Enzyme (OYE;1 EC 1.6.99.1) is an NADPH oxidoreductase that contains flavin mononucleotide (FMN) as the prosthetic group. OYE was initially isolated from brewer's bottom yeast (1, 2) and was the first enzyme in which a vitaminderived molecule responsible for catalysis was shown to be associated with the protein in a 1:1 stoichiometry (3, 4). The protein was able to oxidize NADPH and, somewhat less efficiently, NADH (5, 6). The physiological oxidant of OYE has yet to be determined, but oxygen and a number of quinones can act in this capacity to complete the redox cycle (7). More recently it was found that 2-cyclohexenone (8) and a large number of other ␣,␤-unsaturated aldehydes and ketones are able to act as effective electron acceptors (9), suggesting that the physiological function of the enzyme might be the reduction of such a compound.Phenolic ligands bind to Old Yellow Enzyme with perturbation of the flavoprotein spectra and formation of striking long wavelength (500 -800 nm) absorbance bands (6, 10). This binding is affected by substituents on the phenol and a correlation between the energy of the charge-transfer absorption, and the Hammett para constant has been demonstrated. This correlation and an associated one with the redox potential of the flavin (11) have been used as evidence that the phenolate ion is the charge-transfer donor in the enzyme-phenol complex and the isoalloxazine ring of FMN is the acceptor (5, 12).OYE is...

Crystal structure of 12-oxophytodienoate reductase 3 from tomato: Self-inhibition by dimerization

“…3a and 6). Above the FMN, the active-site amino acids His-185 and His-190 activate the C ␣ -C ␤ double bond of the substrate by forming strong hydrogen bonds with the carbonyl oxygen of the substrate (20,23). As shown for OYE, the activated substrate is reduced by hydride transfer from FMNH Ϫ N (5) to C ␤ and protonated at C ␣ by 24).…”

Old yellow enzyme: Reduction of nitrate esters, glycerin trinitrate, and propylene 1,2-dinitrate

“…His-191 plays a role in hydrogen bonding with the ligand oxygen atom or nitro groups, facilitating the orientation of the substrate in the active site so that the site of hydride transfer is positioned over the reactive N5 locus of the flavin (4)(5)(6)(7)(8). The replacement of His-191 by asparagine also has profound effects on the reaction of the reduced mutant enzyme with GTN, as shown in Table 2.…”

“…Wild-type and mutant enzyme forms were prepared as described (2,5,7). NADPH, NADP, glucose 6-phosphate, and glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides) were from Sigma.…”

“…The long wavelength-absorbance band arises from parallel stacking interaction between the phenolate anion and the flavin. Several mutant forms of OYE have been constructed and have confirmed that , and Thr-37 residues interact in ligand binding and catalysis (5)(6)(7). We have discovered that this enzyme catalyzes the reduction of the olefinic bond of ␣,␤-unsaturated carbonyl compounds by using NADPH as a reductant (2,8) and also catalyzes slow aromatization of cyclic enones like cyclohexenone by a novel dismutation reaction (8).…”

Sequential Enzymatic Conversion of α‐Angelica Lactone to γ‐Valerolactone through Hydride‐Independent C=C Bond Isomerization

“…Similarly, the suggested anionic aromatic intermediate would strongly interact with oxidized FMN and, in absence of a proton donor in OYE2‐Y197F, cannot (or only slowly) leave the active site, thereby preventing product formation. In contrast, the reducing activity of variants lacking the catalytic Tyr is not strongly affected (Supporting Information) because the nonaromatic intermediate is easily released and further protonated by the solvent (water) 2a. The significantly lower conversion level at pH 6 (Table 2, entry 2) strongly supports the involvement of a base in the deprotonation step.…”