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...

Arginine methylation is a post-translational modification that impacts gene expression in both the cytoplasm and nucleus. Here, we demonstrate that arginine methylation also affects mitochondrial gene expression in the protozoan parasite, Trypanosoma brucei. Down-regulation of the major trypanosome type I protein arginine methyltransferase, TbPRMT1, leads to destabilization of specific mitochondrial mRNAs. We provide evidence that some of these effects are mediated by the mitochondrial RNAbinding protein, RBP16, which we previously demonstrated affects both RNA editing and stability. TbPRMT1 catalyzes methylation of RBP16 in vitro. Further, MALDI-TOF-MS analysis of RBP16 isolated from TbPRMT1-depleted cells indicates that, in vivo, TbPRMT1 modifies two of the three known methylated arginine residues in RBP16. Expression of mutated, nonmethylatable RBP16 in T. brucei has a dominant negative effect, leading to destabilization of a subset of those mRNAs affected by TbPRMT1 depletion. Our results suggest that the specificity and multifunctional nature of RBP16 are due, at least in part, to the presence of differentially methylated forms of the protein. However, some effects of TbPRMT1 depletion on mitochondrial gene expression cannot be accounted for by RBP16 action. Thus, these data implicate additional, unknown methylproteins in mitochondrial gene regulation.

Mitochondrial gene expression in Trypanosoma bruceiinvolves the coordination of multiple events including polycistronic transcript cleavage, polyadenylation, RNA stability, and RNA editing. Arg methylation of RNA binding proteins has the potential to influence many of these processes via regulation of protein-protein and protein-RNA interactions. Here we demonstrate that Arg methylation differentially regulates the RNA binding capacity and macromolecular interactions of the mitochondrial gene regulatory protein, RBP16. We show that, in T. brucei mitochondria, RBP16 forms two major stable complexes: a 5 S multiprotein complex and an 11 S complex consisting of the 5 S complex associated with guide RNA (gRNA). Expression of a non-methylatable RBP16 mutant protein demonstrates that Arg methylation of RBP16 is required to maintain the proteinprotein interactions necessary for assembly and/or stability of both complexes. Down-regulation of the major trypanosome type 1 protein arginine methyltransferase, TbPRMT1, disrupts formation of both the 5 and 11 S complexes, indicating that TbPRMT1-catalyzed methylation of RBP16 Arg-78 and Arg-85 is critical for complex formation. We also show that Arg methylation decreases the capacity of RBP16 to associate with gRNA. This is not a general effect on RBP16 RNA binding, however, since methylation conversely increases the association of the protein with mRNA. Thus, TbPRMT1-catalyzed Arg methylation has distinct effects on RBP16 gRNA and mRNA association and gRNA-containing ribonucleoprotein complex (gRNP) formation.Gene regulation in kinetoplastid protozoa such as trypanosomes and Leishmania is effected primarily at the post-transcriptional level (1). In the mitochondria of Trypanosoma brucei, gene expression involves the coordination of multiple processes including ribonucleolytic processing of primary transcripts, polyadenylation, RNA stability, and RNA editing (2-5).2 RNA editing is required for the creation of translatable mRNAs from 12 of the 18 mitochondrial protein-coding genes. Kinetoplastid editing entails insertion and deletion of uridine residues at numerous editing sites as directed by mitochondrially encoded, trans-acting guide RNAs (gRNAs). 3 During editing, the gRNA 5Ј anchor region base pairs with the target mRNA 3Ј of the editing site. An internal information coding region, present within the gRNA, guides uridine insertion and deletion through base pairing. A post-transcriptionally added poly(U) tail, present on the 3Ј end of all mature gRNAs, provides stabilization of mRNA-gRNA interactions. This transcript maturation progresses in a 3Ј-5Ј direction and is catalyzed by a large multisubunit complex termed the editosome (4, 6 -8). The enzymatic reactions involved in editing include endonucleolytic cleavage of the mRNA, uridine insertion or deletion, and religation of the 3Ј and 5Ј mRNA fragments. Although the core components of the editosome are under intense study, accessory proteins that augment and/or regulate editing have only just begun to be identified (9, 10...

α-Dioxygenases (α-DOX) oxygenate fatty acids into 2R-hydroperoxides. Despite low sequence identity, α-DOX share common catalytic features with Cyclooxygenases (COX), including the use of a tyrosyl radical during catalysis. We determined the x-ray crystal structure of Arabidopsis thaliana α-DOX to 1.5Å resolution. The α-DOX structure is monomeric, predominantly α-helical and comprised of two domains. The base domain exhibits low structural homology with the membrane-binding domain of COX but lies in a similar position with respect to the catalytic domain. The catalytic domain shows the highest similarity with the COX catalytic domain, where 21 of the 22 α-helical elements are conserved. Helices H2, H6, H8, and H17 form the heme binding cleft and walls of the active site channel. His-318, Thr-323, and Arg-566 are located near the catalytic tyrosine, Tyr-386, at the apex of the channel, where they interact with a chloride ion. Substitutions at these positions, coupled with kinetic analyses confirm previous hypotheses that implicate these residues as being involved in binding and orienting the carboxylate group of the fatty acid for optimal catalysis. Unique to α-DOX is the presence of two extended inserts on the surface of the enzyme that restrict access to the distal face of the heme, providing an explanation for the observed reduced peroxidase activity of the enzyme. The α-DOX structure represents the first member of the α-DOX subfamily to be structurally characterized within the cyclooxygenase-peroxidase family of heme-containing proteins.