Substrate Recognition by “Password” in <i>p</i>-Hydroxybenzoate Hydroxylase

Palfey, Bruce A.; Moran, Graham R.; Entsch, Barrie; Ballou, David P.; Massey, Vincent

doi:10.1021/bi9826613

Cited by 93 publications

(146 citation statements)

References 25 publications

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“…This spin-allowed step is ratelimiting in several flavin-oxidases and -oxygenases (59). Deprotonation of both the flavin and, in monooxygenases, the organic substrate occurs, the latter in a base-catalyzed manner (35,36,60). The presence of an active site positive charge lowers the height of the activation barrier in some well studied flavoproteins (37) by stabilizing the superoxide anion and helping to minimize the electron transfer reorganization energy () (38,39).…”

Section: Resultsmentioning

confidence: 99%

Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Machovina

Usselman

DuBois

2016

Journal of Biological Chemistry

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Members of the antibiotic biosynthesis monooxygenase family catalyze O 2 -dependent oxidations and oxygenations in the absence of any metallo-or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O 2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater. As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pK a . The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O 2 -dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O 2 reactivity via markedly different means.

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Section: Resultsmentioning

confidence: 99%

Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Machovina

Usselman

DuBois

2016

Journal of Biological Chemistry

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“…According to the structural model of HbpA (18) (39,40). In p-hydroxybenzoate hydroxylase, Tyr 201 is critically involved in the ionization of the substrate, thus affecting flavin movement and allowing efficient hydroxylation (41,42). The substitution Y201F results in an increased uncoupling of NADH oxidation from substrate hydroxylation up to 95% (43).…”

Section: Discussionmentioning

confidence: 99%

Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

Meyer

Würsten

Schmid

et al. 2002

Journal of Biological Chemistry

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“…The deprotonation of pOHB is necessary for facilitating the hydroxylation process because the hydroxylation rate constant increases ϳ6-fold with a rise in pH according to a pK a of 7.1 (9, 13). The pK a of pOHB bound in PHBH is lowered by a hydrogen-bonding network that links pOHB with Tyr-201, Tyr-385, molecular water, and the surface residue His-72 (6,9,13,14,40). Recently, a study of styrene monooxygenase, another two-component flavin-dependent enzyme that also employs C4a-hydroperoxy flavin as an oxygenation species, has shown that the oxygenation (epoxidation) reaction of styrene monooxygenase is favored at lower pH values, consistent with a pK a of 7.7 (41).…”

Section: ⅐Hpamentioning

confidence: 99%

“…For the PHBH reaction, the hydroxylation rate constant increases upon an increase in pH with a pK a of 7.1 (13). This pK a value corresponds to the pK a of His-72, which is located on the protein surface and controls the H-bond network that facilitates removal of the phenolic proton of pOHB (9,13,14). For two-component flavin-dependent monooxygenases, the mode of oxygen atom transfer in hydroxylation of aromatic compounds has never been explored.…”

mentioning

confidence: 99%

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

Tongsook

Sucharitakul

Thotsaporn

et al. 2011

Journal of Biological Chemistry

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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 *

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Substrate Recognition by “Password” in p-Hydroxybenzoate Hydroxylase

Cited by 93 publications

References 25 publications

Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations

Hydroxylation of Indole by Laboratory-evolved 2-Hydroxybiphenyl 3-Monooxygenase

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

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