Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function

Liu, W.; Wang, L. H.; Fabian, Piotr; Hayashi, Yuka; McGinley, Chris M.; Donk, Wilfred A. van der; Kulmacz, Richard J.

doi:10.1016/j.plaphy.2006.06.013

Cited by 14 publications

(41 citation statements)

References 23 publications

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“…PIOX and the COX enzymes share sequence similarity of ϳ20% (3,5,15). Analysis of the sequence alignment between O. sativa PIOX and O. aries COX-1 indicates that significant sequence similarities exist between residues 123-609 of PIOX and residues 135-584 of COX-1, which corresponds to the catalytic domain of the COX enzyme (17).…”

Section: Structure Prediction and Identification Of Candidate Residuementioning

confidence: 99%

“…For this study we chose 18 different fatty acids that were between 14 and 20 carbons in length and that also varied in both the type and number of unsaturated bonds within the fatty acid. The panel of substrates comprised above was generated to also include a subset of fatty acids utilized in an analogous experiment that was performed with A. thaliana PIOX (15), so that comparisons could be made between the two enzymes with respect to their substrate preferences. We also included 5 novel 18-carbon substrates in our panel that have modifications at either the carboxylate group, carbon-1, or carbon-2 (Table 3).…”

Section: F N L I Y R W H C P P Y T T S R a I L S D A Numbering Based mentioning

confidence: 99%

“…The relative activities for A. thaliana PIOX are taken from Ref. 15 and listed here after re-normalizing the values to the oxygenase activity reported for LA in their study. (Fig.…”

Section: F N L I Y R W H C P P Y T T S R a I L S D A Numbering Based mentioning

confidence: 99%

“…PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6, 7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3, 10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous decarboxylation to shorter aldehydes and fatty acids (11,14).…”

mentioning

confidence: 99%

“…PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous decarboxylation to shorter aldehydes and fatty acids (11,14).…”

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confidence: 99%

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His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Koszelak-Rosenblum

Krol

Simmons

et al. 2008

Journal of Biological Chemistry

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Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid ␣-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in K m , whereas no substantial change in K m was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/ R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14 -20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis.Pathogen attack on plants brings about the activation of multiple enzyme systems that results in the production of oxylipins from 18 -22 carbon fatty acid precursors. The generation of these bioactive lipid mediators initiates and sustains the defense reaction of the plant against insects, bacteria, fungi, and other pathogens (1, 2). One of the enzymes up-regulated during the host defense response is pathogen-inducible oxygenase (PIOX), 2 which catalyzes a non-lipoxygenase type of fatty acid oxygenation (3). PIOX belongs to a larger family of heme-containing proteins that oxygenate fatty acids (4), which include the mammalian cyclooxygenases (COX-1 and COX-2; (5)), linoleate diol synthase (LDS) from the fungus Gaeumannomyces graminis (6, 7), and a Pseudomonas alcalignes protein of unknown function encoded by OrfX (8). PIOX has also been identified in many plant species, including Nicotiana attenuata (9), Nicotiana tabacum (3), Arabidopsis thaliana (3, 10), O. sativa (11), Capsicum annuum (12), and Lycopersicon esculentum (13).PIOX utilizes stereoselective oxygenation to convert linoleic acid (LA) (18:2, n-6) and other fatty acid substrates to their corresponding 2R-hydroperoxides, generating a novel class of oxylipins (3,11,14,15). The resulting 2R-hydroperoxides undergo spontaneous deca...

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Section: Structure Prediction and Identification Of Candidate Residuementioning

confidence: 99%

Section: F N L I Y R W H C P P Y T T S R a I L S D A Numbering Based mentioning

confidence: 99%

“…The relative activities for A. thaliana PIOX are taken from Ref. 15 and listed here after re-normalizing the values to the oxygenase activity reported for LA in their study. (Fig.…”

Section: F N L I Y R W H C P P Y T T S R a I L S D A Numbering Based mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

Koszelak-Rosenblum

Krol

Simmons

et al. 2008

Journal of Biological Chemistry

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α‐Dioxygenases (α‐DOXs): Promising Biocatalysts for the Environmentally Friendly Production of Aroma Compounds

et al. 2022

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Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco‐friendly synthetic routes is in demand. α‐Dioxygenases (α‐DOXs) are heme‐dependent oxidative enzymes biologically involved in the initial step of plant FA α‐oxidation during which molecular oxygen is incorporated into the Cα‐position of a FA (Cn) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn‐1). α‐DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α‐DOXs with emphasis on newly discovered cyanobacterial α‐DOXs as well as analytical methods to measure α‐DOX activity in vitro and in vivo.

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Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

2013

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a-Dioxygenases (a-DOX) are heme-containing enzymes found predominantly in plants and fungi, where they generate oxylipins in response to pathogen attack. a-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 a-carbon by a tyrosyl radical generated via the oxidation of the heme moiety by hydrogen peroxide (H 2 O 2 ). We determined the X-ray crystal structures of wild type a-DOX from Oryza sativa, the wild type enzyme in complex with H 2 O 2 , and the catalytically inactive Y379F mutant in complex with the fatty acid palmitic acid (PA). PA binds within the active site cleft of a-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 H 2 O 2 structures provides insight into enzyme activation. The binding of H 2 O 2 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 Od2 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.

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Arabidopsis thaliana fatty acid alpha-dioxygenase-1: evaluation of substrates, inhibitors and amino-terminal function

Cited by 14 publications

References 23 publications

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

His-311 and Arg-559 Are Key Residues Involved in Fatty Acid Oxygenation in Pathogen-inducible Oxygenase

α‐Dioxygenases (α‐DOXs): Promising Biocatalysts for the Environmentally Friendly Production of Aroma Compounds

Crystal structures of α‐dioxygenase from Oryza sativa: Insights into substrate binding and activation by hydrogen peroxide

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