Dataset Information

Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.

ABSTRACT: Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O₂. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over ?12 Å and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe(III)-superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (?_max = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of ¹⁷O₂ (I = ⁵/₂) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- and multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release ?300-fold, providing insight into the regulatory mechanism of ROs.

SUBMITTER: Rogers MS

PROVIDER: S-EPMC6856394 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.

Rogers Melanie S MS Lipscomb John D JD

Biochemistry 20190515 52

Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that <i>cis</i>-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. He ...[more]

PMID: 31066545

Similar Datasets

Project description:Rieske nonheme iron oxygenases (ROs) catalyze the oxidation of a wide variety of substrates and play important roles in aromatic compound degradation and polycyclic aromatic hydrocarbon degradation. Those Rieske dioxygenases that usually act on hydrophobic substrates have been extensively studied and structurally characterized. Here, we report the crystal structure of a novel Rieske monooxygenase, NagGH, the oxygenase component of a salicylate 5-monooxygenase from Ralstonia sp. strain U2 that catalyzes the hydroxylation of a hydrophilic substrate salicylate (2-hydroxybenzoate), forming gentisate (2, 5-dihydroxybenzoate). The large subunit NagG and small subunit NagH share the same fold as that for their counterparts of Rieske dioxygenases and assemble the same α3β3 hexamer, despite that they share low (or no identity for NagH) sequence identities with these dioxygenase counterparts. A potential substrate-binding pocket was observed in the vicinity of the nonheme iron site. It featured a positively charged residue Arg323 that was surrounded by hydrophobic residues. The shift of nonheme iron atom caused by residue Leu228 disrupted the usual substrate pocket observed in other ROs. Residue Asn218 at the usual substrate pocket observed in other ROs was likewise involved in substrate binding and oxidation, yet residues Gln316 and Ser367, away from the usual substrate pocket of other ROs, were shown to play a more important role in substrate oxidation than Asn218. The unique binding pocket and unusual substrate-protein hydrophilic interaction provide new insights into Rieske monooxygenases.IMPORTANCE Rieske oxygenases are involved in the degradation of various aromatic compounds. These dioxygenases usually carry out hydroxylation of hydrophobic aromatic compounds and supply substrates with hydroxyl groups for extradiol/intradiol dioxygenases to cleave rings, and have been extensively studied. Salicylate 5-hydroxylase NagGH is a novel Rieske monooxygenase with high similarity to Rieske dioxygenases, and also shares reductase and ferredoxin similarity with a Rieske dioxygenase naphthalene 1,2-dioxygenase (NagAcAd) in Ralstonia sp. strain U2. The structure of NagGH, the oxygenase component of salicylate 5-monooxygenase, gives a representative of those monooxygenases and will help us understand the mechanism of their substrate binding and product regio-selectivity.

Project description:Phenylalanine hydroxylase (PheH) and tryptophan hydroxylase (TrpH) catalyze the aromatic hydroxylation of phenylalanine and tryptophan, forming tyrosine and 5-hydroxytryptophan, respectively. The reactions of PheH and TrpH have been investigated with [4-(2)H]-, [3,5-(2)H(2)]-, and (2)H(5)-phenylalanine as substrates. All (D)k(cat) values are normal with Delta117PheH, the catalytic core of rat phenylalanine hydroxylase, ranging from 1.12-1.41. In contrast, for Delta117PheH V379D, a mutant protein in which the stoichiometry between tetrahydropterin oxidation and amino acid hydroxylation is altered, the (D)k(cat) value with [4-(2)H]-phenylalanine is 0.92 but is normal with [3,5-(2)H(2)]-phenylalanine. The ratio of tetrahydropterin oxidation to amino acid hydroxylation for Delta117PheH V379D shows a similar inverse isotope effect with [4-(2)H]-phenylalanine. Intramolecular isotope effects, determined from the deuterium contents of the tyrosine formed from [4-(2)H]-and [3,5(2)H(2)]-phenylalanine, are identical for Delta117PheH and Delta117PheH V379D, suggesting that steps subsequent to oxygen addition are unaffected in the mutant protein. The inverse effects are consistent with the reaction of an activated ferryl-oxo species at the para position of the side chain of the amino acid to form a cationic intermediate. The normal effects on the (D)k(cat) value for the wild-type enzyme are attributed to an isotope effect of 5.1 on the tautomerization of a dienone intermediate to tyrosine with a rate constant 6- to7-fold that for hydroxylation. In addition, there is a slight ( approximately 34%) preference for the loss of the hydrogen originally at C4 of phenylalanine. With (2)H(5)-indole-tryptophan as a substrate for Delta117PheH, the (D)k(cat) value is 0.89, consistent with hydroxylation being rate-limiting in this case. When deuterated phenylalanines are used as substrates for TrpH, the (D)k(cat) values are within error of those for Delta117PheH V379D. Overall, these results are consistent with the aromatic amino acid hydroxylases all sharing the same chemical mechanism, but with the isotope effect for hydroxylation by PheH being masked by tautomerization of an enedione intermediate to tyrosine.

Dataset Information

Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.

Publications

Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets