Old Yellow Enzyme: Stepwise reduction of nitro-olefins and catalysis of aci-nitro tautomerization

Abstract

The Old Yellow Enzyme has been shown to catalyze efficiently the NADPH-linked reduction of nitro-olefins. The reduction of the nitro-olefin proceeds in a stepwise fashion, with formation of a nitronate intermediate that is freely dissociable from the enzyme. The first step involves hydride transfer from the enzyme-reduced flavin to carbon 2 of the nitro-olefin. The protonation of the nitronate at carbon 1 to form the final nitroalkane product also is catalyzed by the enzyme and involves Tyr-196 as an active site acid͞base. This residue also is involved in aci-nitro tautomerization of nitroalkanes, the first example of a nonredox reaction catalyzed by the enzyme.O ld Yellow Enzyme (OYE; EC 1.6.99.1), isolated from brewer's bottom yeast, was the first enzyme where the requirement of a prosthetic group (cofactor) for catalysis was demonstrated (1). OYE is a mixture of homodimers and a heterodimer arising from two yeast genes, with each monomer binding a molecule of flavin mononucleotide. Although OYE's exact physiological function is still unknown, many tight binding ligands such as phenols and steroids have been identified, raising the possibility that this enzyme might be involved in yeast sterol metabolism (2). Upon binding with phenols and heteroaromatic compounds containing ionizable hydroxyl groups OYE produces characteristic long wavelength charge transfer complexes (3).The crystal structure of OYE has been solved, revealing several amino acid residues around the active site of the enzyme to effect catalysis and ligand binding (4). Phenolic compounds are held parallel to the flavin by hydrogen bonding of the phenolate oxygen with . The long wavelength absorbance band arises from parallel stacking interaction between the phenolate anion and the flavin. Several mutants of OYE have been constructed and confirmed the importance of , and Thr-37 residues in ligand binding interactions and catalysis (5-7). We also 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). Using R-( 2 H) NADPH in D 2 O it was unequivocally established that reduction of ␣,␤-unsaturated carbonyl compounds proceeds via stereospecific transfer of hydride to the carbon atom ␤ to the carbonyl function and transfer a proton to the ␣ position, resulting in trans addition to the double bond (8). Another class of compounds similar to ␣,␤-unsaturated carbonyls is that of unsaturated nitro compounds. Like a carbonyl group a nitro group exerts a strong electron attracting influence within the molecule, enhancing the acidity of the hydrogen atoms attached to the carbon ␣ to the substituent group. Nitro compounds also exhibit a tautomerism exactly analogous to keto-enol tautomerism. Chiral nitro compounds are an important class of synthetically useful compounds, which can be easily converted into chiral amines, and chiral amines are useful interm...

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

“…In the reaction of OYE1 with other substrates, such as ␣,␤-unsaturated aldehydes and ketones (8) or unsaturated nitro substrates like nitrocyclohexene (9), Tyr-196 acts as active-site acid, providing the second hydrogen atom involved in reduction of the substrate double bond as a proton. In such reactions, the mutant enzyme Y196F is catalytically very much impaired (6,9). In contrast, this enzyme form is quite competent in catalysis of denitration of organic nitrate esters; indeed, as shown in Table 2, the limiting rate constant of oxidation by GTN is twice that with wild-type enzyme, although the K d is increased by an order of magnitude.…”

Modulation of Nitro-fatty Acid Signaling

“…Additionally, the two-electron reduction of non-aromatic nitroalkenes to nitroalkanes occurs in both bacteria and yeast (55,56). In particular, OYE (EC 1.6.99.1) the archetypical member of the old yellow enzyme flavoprotein family, reacts with non-native, non-aromatic nitroalkenes such as nitrocyclohexene, nitrostyrene, and nitrovinylthiophene via NADPH-dependent mechanisms to generate the corresponding nitroalkane by the intermediate formation of a nitronate derivative (32). Finally, mammalian enzymes such as thioredoxin reductase, NAD(P)H:quinone oxidoreductases, NADPH: cytochrome P450 reductase, and ferredoxin:NADP ϩ reductase reduce nitroaromatic compounds either by one-or two-electron transfer to the nitro moiety, yielding the corresponding nitroso, hydroxylamine, or amine derivatives (57)(58)(59)(60)(61).…”

“…However, biosynthesis of other nitroalkene-containing molecules has also been described in Gram-negative bacteria, fungi, plants, and insects (27)(28)(29)(30). Several enzymes capable of nitroalkene reduction have been described in these organisms, including pentaerythritol tetranitrate (PETN) reductase from Enterobacter cloacae PB2 and members of the old yellow enzyme family such as Escherichia coli N-ethylmaleimide reductase, Pseudomonas putida M10 morphinone reductase, and OYE1 from Saccharomyces carlsbergensis (31)(32)(33).…”

‘New uses for an Old Enzyme’ – the Old Yellow Enzyme family of flavoenzymes

“…A reaction of particular interest is the hydrogenation of nitroalkenes, since it involves the intermediate formation of a nitronate, followed by slow protonation of the intermediate to the nitroalkane. Accumulation of the nitronate can be forced by the Y196F mutation of OYE (Meah & Massey, 2000), providing the attractive prospect of biocatalytic production of a nitronate. When integrated with chemical alkylation, this could provide a route to nitroalkanes with two chiral centres.…”