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Crystal structures of ?-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

ABSTRACT: ?-Dioxygenases (?-DOX) are heme-containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. ?-DOX oxygenate a variety of 14-20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro-R hydrogen from the ?-carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H2 O2 ). We determined the X-ray crystal structures of wild type ?-DOX from Oryza sativa, the wild type enzyme in complex with H2 O2 , and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of ?-DOX such that the carboxylate forms ionic interactions with His-311 and Arg-559. Thr-316 aids in the positioning of carbon-2 for hydrogen abstraction. Twenty-five of the twenty eight contacts made between PA and residues lining the active site occur within the carboxylate and first eight carbons, indicating that interactions within this region of the substrate are responsible for governing selectivity. Comparison of the wild type and H2 O2 structures provides insight into enzyme activation. The binding of H2 O2 at the distal face of the heme displaces residues His-157, Asp-158, and Trp-159 ? 2.5 Å from their positions in the wild type structure. As a result, the O?2 atom of Asp-158 interacts with the Ca atom in the calcium binding loop, the side chains of Trp-159 and Trp-213 reorient, and the guanidinium group of Arg-559 is repositioned near Tyr-379, poised to interact with the carboxylate group of the substrate.

SUBMITTER: Zhu G

PROVIDER: S-EPMC3795501 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Crystal structures of α-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

Zhu Guangyu G Koszelak-Rosenblum Mary M Malkowski Michael G MG

Protein science : a publication of the Protein Society 20130904 10

α-Dioxygenases (α-DOX) are heme-containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. α-DOX oxygenate a variety of 14-20 carbon fatty acids containing up to three unsaturated bonds through stereoselective removal of the pro-R hydrogen from the α-carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H2 O2 ). We determined the X-ray crystal structures of wild type α-DOX from Oryza sativa, ...[more]

PMID: 23934749

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Project description:Rieske dioxygenases catalyze the reductive activation of O2 for the formation of cis-dihydrodiols from unactivated aromatic compounds. It is known that O2 is activated at a mononuclear non-heme iron site utilizing electrons supplied by a nearby Rieske iron sulfur cluster. However, it is controversial whether the reactive species is an Fe(III)-(hydro)peroxo or an Fe(II)-(hydro)peroxo (or electronically equivalent species formed by breaking the O-O bond). Here it is shown that benzoate 1,2 dioxygenase oxygenase component (BZDO) prepared in a form with the Rieske cluster oxidized and the mononuclear iron in the Fe(III) state can utilize H2O2 as a source of reduced oxygen to form the correct cis-dihydrodiol product from benzoate. The reaction approaches stoichiometric yield relative to the mononuclear Fe(III) concentration, being limited to a single turnover by inefficient product release from the Fe(III)-product complex. EPR and Mössbauer studies show that the iron remains ferric throughout this single turnover "peroxide shunt" reaction. These results strongly support Fe(III)-(hydro)peroxo (or Fe(V)-oxo-hydroxo) as the reactive species because there is no source of additional reducing equivalents to form the Fe(II)-(hydro)peroxo state. This conclusion could be further tested in the case of BZDO because the peroxide shunt occurs very slowly compared with normal turnover, allowing the reactive intermediate to be trapped for spectroscopic analysis. We attribute the slow reaction rate to a forced change in the normally strict order of the substrate binding and enzyme reduction steps that regulate the catalytic cycle. The reactive intermediate is a high-spin ferric species exhibiting an unusual negative zero field splitting and other EPR and Mössbauer spectroscopic properties reminiscent of previously characterized side-on-bound peroxide adducts of Fe(III) model complexes. If the species in BZDO is a similar adduct, its isomer shift is most consistent with an Fe(III)-hydroperoxo reactive state.

Dataset Information

Crystal structures of ?-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

Publications

Crystal structures of α-dioxygenase from Oryza sativa: insights into substrate binding and activation by hydrogen peroxide.

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