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

Abstract

Rieske non-heme iron oxygenases (ROs) catalyze the oxidation of a wide variety of substrates and play important roles in aromatic compound degradation along with polycyclic aromatic hydrocarbons 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 though they share low (even none for NagH) sequence identities with those dioxygenase counterparts. A potential substrate binding pocket was observed in the vicinity of non-heme iron site. It featured a positively-charged residue Arg323 that was surrounded by hydrophobic residues. The shift of non-heme 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 exactly involved in substrate binding and oxidation, yet residues Gln316 and Ser367 away from that usual substrate pocket of other ROs played a more important role in substrate oxidation than Asn218. The unique binding pocket and unusual substrate-protein hydrophilic interaction provided new insight into Rieske monooxygenases. IMPORTANCE Rieske oxygenases are involved in the degradation of various aromatic compounds. Those dioxygenases, usually carry out hydroxylation of hydrophobic aromatic compounds and supply substrates with hydroxyl groups for extradiol/intradiol dioxygenases to cleave ring, have been extensively studied. Salicylate 5-hydroxylase NagGH is a novel Rieske monooxygenase with high similarity with Rieske dioxygenases, also shares reductase and ferredoxin 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 regioselectivity.