Reaction of 2-Methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) Oxygenase with<i>N</i>-Methyl-5-hydroxynicotinic acid:  Studies on the Mode of Binding, and Protonation Status of the Substrate

Pakotiprapha

et al. 2013

Self Cite

Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein hydroxylase catalyzing the para-hydroxylation of 3-hydroxybenzoate. Results: The oxidative half-reaction was studied using transient kinetics. The enzyme reaction mechanism was elucidated. Conclusion:The product release and the hydroxylation limit the turnovers and the enzyme shows characteristics different from the ortho-hydroxylation enzymes. Significance: This is the first report on the oxygenation mechanism of a para-hydroxylating flavoenzyme.

Section: Reaction Of Reduced Enzyme-3hb Complex With Oxygen In the Prmentioning

confidence: 99%

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

Pakotiprapha

et al. 2013

Self Cite

“…(MHPCO) (33,34), 2-hydroxybiphenyl 3-monooxygenase (35), and kynurenine 3-monooxygenase (36). For two-component monooxygenases, this intermediate was detected in the reactions of bacterial luciferase (37), p-hydroxyphenylacetate hydroxylase (38 -40), chorophenol 4-monooxygenase (41), styrene monooxygenase (42), alkane sulfonate monooxygenase (18), and the oxygenases involved in the biosyntheses of rebeccamycin (43) and actinorhodin (44).…”

Section: ؊1 Smentioning

confidence: 99%

“…Both C4a-hydroperoxy-FMN and C4a-hydroxy-FMN were nonfluorescent, even when various substrate analogs were used (data not shown). In this respect, C 2 is quite different from many flavin-dependent monooxygenases such as HpaA (40), actinorhodin (44), PHBH (51), phenol hydroxylase (28), and 2-methyl-3-hydroxypyridine-5-carboxylic oxygenase (33,34) in which the C4a-hydroxy-flavin is more fluorescent than the C4a-hydroperoxy-flavin. Rate constants of the product formation obtained from rapid-quench experiments also indicate that the hydroxylation occurs via a direct participation of the C4a-hydroperoxy-flavin intermediate, as the product formation occurs prior to the flavin oxidation step.…”

mentioning

confidence: 99%

“…7, step e). For single-component monooxygenases, an uncoupling pathway, resulting in H 2 O 2 generation, is quite common and usually accounts for ϳ5-30% of the amount of a reducing equivalent (NAD(P)H) consumed (3,5,33,34). It is likely that C 2 achieves this hydroxylation efficiency through optimum interactions among HPA, C4a-hydroperoxy-FMN, and the active site residues.…”

mentioning

confidence: 99%

See 1 more Smart Citation

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Ruangchan

et al. 2011

Self Cite

Flavin-dependent monooxygenases catalyze the incorporation of a single atom of molecular oxygen into organic substrates (1, 2). The enzymes have been classified into six classes according to their catalytic and structural properties. They have also been categorized into two major types according to their protein components: a single-component type, in which reduction of a flavin cofactor and oxygenation of an organic substrate occurs within the same single polypeptide chain; and a two-component type, in which each reaction occurs on separate proteins (1, 2). Single-component monooxygenases have been identified since the 1960s and have been found to be involved in the aerobic metabolism of aromatic and aliphatic compounds in various organisms (2-4). The well known prototype for single-component monooxygenases is p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens (5, 6). The first enzyme identified as a two-component monooxygenase was bacterial luciferase (7). Nevertheless, most of the two-component monooxygenases have been identified only during the past decade and have increasingly emerged as common enzymes in nature that are involved in many important reactions in various microorganisms (1,8). Reactions catalyzed by two-component monooxygenases include oxygenation and halogenation of organic compounds such as p-hydroxyphenylacetate (9), phenol (10), trichorophenol (11, 12), p-nitrophenol (13), styrene (14 -16), alkane sulfonate (17, 18), and EDTA (19). Two-component monooxygenases are also involved in oxygenation and halogenation reactions in the biosynthetic pathways of actinorhodin (ActVA) (20), angucyclin (21), enediyne (SgcC) (22), rebeccamycin (RebH) (23), pyrrolnitrin (PrnA) (24), violacein (25), kutzneride (26), and differentiation-inducing factor-1 (27).All flavin-dependent monooxygenases perform oxygenation through the participation of a reactive intermediate, C4a-hydroperoxy-flavin, which has been well documented and detected by transient kinetics for the reactions of singlecomponent monooxygenases. These monooxygenases include p-hydroxybenzoate hydroxylase (PHBH) 3 (5, 6), phenol hydroxylase (28), melilotate hydroxylase (29), antranilate hydroxylase (30), Baeyer-Villiger monooxygenases (31, 32), 2-methyl-3-hydroxypyridine-5-carboxylic oxygenase * This work was supported by Grants BRG5180002 (to P. C.) and MRG5380240 (to J. S.) from the Thailand Research Fund and a grant from the Faculty of Science, Mahidol University (to P. C.

“…Selective binding of a deprotonated hydroxyl group to facilitate the electrophilic aromatic substitution has been observed for the reaction of MHPCO (11,12,39). Although the substrate (MHPC) of MHPCO exists as a tautomeric mixture at pH 7.0, only the tripolar ionic form in which the hydroxyl group (equivalent to the phenolic group in HPA) is deprotonated binds to MHPCO (12,39). For the PHBH reaction, both the phenolic and phenolate forms of the substrate (pOHB) can bind to the enzyme, depending on the pH of the solution.…”

Section: ⅐Hpamentioning

confidence: 99%

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

Thotsaporn

et al. 2011

Self Cite

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 *