Post-translational lipidation provides critical modulation of the functions of some proteins. Isoprenoids (i.e., farnesyl or geranylgeranyl groups) are attached to cysteine residues in proteins containing C-terminal CaaX sequence motifs. Isoprenylation is followed by cleavage of the aaX amino acid residues and, in some cases, by additional proteolytic cuts. We determined the crystal structure of the CaaX protease Ste24p, a zinc metalloprotease catalyzing two proteolytic steps in the maturation of yeast mating pheromone a-factor. The Ste24p core structure is a ring of seven transmembrane helices enclosing a voluminous cavity containing the active-site and substrate binding groove. The cavity is accessible to the external milieu via gaps between splayed transmembrane helices. We hypothesize that cleavage proceeds via a processive mechanism of substrate insertion, translocation and ejection.

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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