Interactions with the Substrate Phenolic Group Are Essential for Hydroxylation by the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Abstract

plex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ϳ70% over a pH range of 6 -10, whereas product formation for S146C decreased from ϳ65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the His ␦؉ ⅐HPA ␦؊ complex needed for hydroxylation.Incorporation of single oxygen atoms into organic compounds (monooxygenation) by hydroxylation or epoxidation is an important biological process for aerobic organisms. The monooxygenation reaction is generally catalyzed by cytochrome P450, metalloenzymes, pterin-dependent and flavindependent monooxygenases (1). Flavin-dependent monooxygenases are involved in a wide variety of biological processes (2, 3). These enzymes catalyze the monooxygenation of many aromatic and aliphatic compounds (3).Based on the number of proteins required for catalysis, flavin-dependent monooxygenases can be classified into singleprotein component and two-protein component types (2-5). Besides an organic substrate, both types of enzymes require NAD(P)H and oxygen as co-substrates. The initial part of the reaction is the reduction of an enzyme-bound flavin by NAD(P)H to generate reduced flavin followed by the reaction of reduced flavin with oxygen to form the C4a-(hydro)peroxy flavin, a key intermediate that is required for oxygenation of an organic substrate. Oxygenation occurs via an oxygen atom transfer from C4a-hydroperoxy flavin to an organic substrate, resulting in the formation of a C4a-hydroxy flavin intermediate and an oxygenated product. The C4a-hydroxy flavin dehydrates at the last step of the reaction to form the final species, oxidized flavin (2-4, 6). All steps for the reactions of singleprotein component flavin-dependent monooxygenases occur within the same polypeptide, whereas for the two-protein component type the flavin reduction occurs on a reductase component and the oxygenation occurs on an oxygenase component (4 -8). The mechanism by which the reduced flavin is transferred involves simple diffusion or protein-protein contacts (5,8). Although single-component flavin-dependent monooxygenases have been studied for more than 40 years, two-component flavin-dependent monooxygenases have only received significant attention during the past decade after recent discoveries of their involvement in a wide variety of reactions (2, 5).The mechanism by which the oxygen atom transfer occurs in flavin-dependent monooxygenases is well understood for only a few enzymatic systems. The best understood oxygenation reaction is the hydroxylation of aromatic compounds catalyzed *

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle

“…An exception to this was found in the reaction of phenol hydroxylase (27) in which the enzyme showed formation of C4a-peroxyflavin anion before protonation to C4a-hydroperoxyflavin. The latter species is required in these aromatic hydroxylases because their reactions are involved in electrophilic aromatic substitution in which the C4a-hydroperoxyflavin acts as an electrophile (4,7,9). Although the advantage of having discrete steps of C4a-peroxyflavin and C4a-hydroperoxyflavin formation in 3HB6H and phenol hydroxylase is not clear, it highlights the differences in the proton transfer pathways among flavin-dependent hydroxylases.…”

The substrate oxidation mechanism of pyranose 2‐oxidase and other related enzymes in the glucose–methanol–choline superfamily

“…It is still not known whether C4a‐(hydro)peroxyflavins are common among other flavoenzyme oxidases as well, but the other enzymes simply fail to stabilize the intermediate, or whether this intermediate is unique to P2O . The C4a‐hydroperoxyflavin intermediate is common for flavoprotein monooxygenases, because it is required as an oxygenating reagent . However, for P2O, the C4a‐hydroperoxyflavin merely eliminates H 2 O 2 to generate oxidized FAD (Scheme ; Table ) .…”

The C-terminal Domain of 4-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii Is an Autoinhibitory Domain

“…HPAH belongs to a large family of two-component flavin-dependent monooxygenases. Catalytic and structural studies of the enzymes from several bacterial species, including Pseudomonas putida (16,17), Pseudomonas aeruginosa (15), Escherichia coli (18), Klebsiella pneumonia (19), Sulfolobus tokodaii (20), Thermus thermophilus (21,22), and A. baumannii (12,(23)(24)(25)(26)(27)(28)(29)(30)(31), have been carried out. HPAH consists of a reductase component (C 1 ) that catalyzes reduction of FMN to generate FMNH Ϫ for its oxygenase (C 2 ), which catalyzes the oxygenation of HPA (12,23).…”