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Structural and Biochemical Analysis Reveals a Distinct Catalytic Site of Salicylate 5-Monooxygenase NagGH from Rieske Dioxygenases.

ABSTRACT: 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.

SUBMITTER: Hou YJ

PROVIDER: S-EPMC8105011 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Hexachlorobenzene (HCB), as one of the persistent organic pollutants (POPs) and a possible human carcinogen, is especially resistant to biodegradation. In this study, HcbA1A3, a distinct flavin-N5-peroxide-utilizing enzyme and the sole known naturally occurring aerobic HCB dechlorinase, was biochemically characterized. Its apparent preference for HCB in binding affinity revealed that HcbA1 could oxidize only HCB rather than less-chlorinated benzenes such as pentachlorobenzene and tetrachlorobenzenes. In addition, the crystal structure of HcbA1 and its complex with flavin mononucleotide (FMN) were resolved, revealing HcbA1 to be a new member of the bacterial luciferase-like family. A much smaller substrate-binding pocket of HcbA1 than is seen with its close homologues suggests a requirement of limited space for catalysis. In the active center, Tyr362 and Asp315 are necessary in maintaining the normal conformation of HcbA1, while Arg311, Arg314, Phe10, Val59, and Met12 are pivotal for the substrate affinity. They are supposed to place HCB at a productive orientation through multiple interactions. His17, with its close contact with the site of oxidation of HCB, probably fixes the target chlorine atom and stabilizes reaction intermediates. The enzymatic characteristics and crystal structures reported here provide new insights into the substrate specificity and catalytic mechanism of HcbA1, which paves the way for its rational engineering and application in the bioremediation of HCB-polluted environments.IMPORTANCE As an endocrine disrupter and possible carcinogen to human beings, hexachlorobenzene (HCB) is especially resistant to biodegradation, largely due to difficulty in its dechlorination. The lack of knowledge of HCB dechlorinases limits their application in bioremediation. Recently, an HCB monooxygenase, HcbA1A3, representing the only naturally occurring aerobic HCB dechlorinase known so far, was reported. Here, we report its biochemical and structural characterization, providing new insights into its substrate selectivity and catalytic mechanism. This research also increases our understanding of HCB dechlorinases and flavin-N5-peroxide-utilizing enzymes.

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Structural and Biochemical Analysis Reveals a Distinct Catalytic Site of Salicylate 5-Monooxygenase NagGH from Rieske Dioxygenases.

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