Tomato CYP74C3 is a Multifunctional Enzyme not only Synthesizing Allene Oxide but also Catalyzing its Hydrolysis and Cyclization

The mechanism of the recombinant tomato allene oxide synthase (LeAOS3, CYP74C3) was studied. Incubations of linoleic acid (9S)-hydroperoxide with dilute suspensions of LeAOS3 (10-20 s, 0 °C) yield mostly the expected allene oxide (12Z)-9,10-epoxy-10,12-octadecadienoic acid (9,10-EOD), which was detected as its methanol-trapping product. In contrast, the relative yield of 9,10-EOD progressively decreased when the incubations were performed with fourfold, tenfold, or 80-fold larger amounts of LeAOS3, while -ketol and the cyclopentenone rac-cis-10-oxo-11-phytoenoic acid (10-oxo-PEA) became the predominant products. Both the -ketol and 10-oxo-PEA were also produced when LeAOS3 was exposed to preformed 9,10-EOD, which was generated by maize allene oxide synthase (CYP74A). LeAOS3 also converted linoleic acid (13S)-hydroperoxide into the corresponding allene oxide, but with about tenfold lower yield of cyclopentenone. The results indicate that in contrast to the ordinary allene oxide synthases (CYP74A subfamily), LeAOS3 (CYP74C subfamily) is a multifunctional enzyme, catalyzing not only the synthesis, but also the hydrolysis and cyclization of allene oxide.

Keywords: allene oxide synthase • enzyme catalysis • metabolism • oxylipins • tomato

Source: ChemBioChem (2008) Vol. 9, Issue 15, p. 2498 - 2505

Determinants governing the CYP74 catalysis: Conversion of allene oxide synthase into hydroperoxide lyase by site-directed mutagenesis

Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the “I-helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

Keywords: Cytochrome P450; CYP74 family; Allene oxide synthase; Hydroperoxide lyase; Site-directed mutagenesis

Source: FEBS Letters (2008) Vol. 582, Issues 23-24, Pages 3423-3428

My Article on BKCS 2009

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

Bulletin of the Korean Chemical Society. (2009) vol 30, pages 719-723

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Biosynthesis and Metabolism of Jasmonates

Jasmonates are derived from oxygenated fatty acids via the octadecanoid pathway and characterized by a pentacyclic ring structure. They have regulatory function as signaling molecules in plant development and adaptation to environmental stress. Until recently, it was the cyclopentanone jasmonic acid (JA) that attracted most attention as a plant growth regulator. It becomes increasingly clear, however, that biological activity is not limited to JA but extends to, and may even differ between its many metabolites and conjugates as well as its cyclopentenone precursors. The enzymes of jasmonate biosynthesis and metabolism may thus have a regulatory function in controlling the activity and relative levels of different signaling molecules. Such a function is supported by both the characterization of loss of function mutants in Arabidopsis, and the biochemical characterization of the enzymes themselves.

Source: Journal of Plant Growth Regulation (2005) vol. 23, p. 179-199

Jasmonate-Responsive Gene Expression

Jasmonic acid (JA) and its volatile methyl ester (MeJA) belong to a family of lipid-derived signalling molecules that affect many aspects of plant life, including defence against certain pathogens and insects and some developmental processes. JA signal transduction leads to modulation of the expression of primary response genes, the products of which lead to the expression of secondary response genes. The ORCA3 transcription factor from Catharanthus roseus is a good candidate for a terminal component of the JA signal transduction pathway. To our knowledge, not a single component of the primary JA signal transduction pathway has been characterized to date in Arabidopsis. Many transcriptional components of secondary JA response pathways have been described in this model plant species, and are reviewed here. Our review advocates a strong adherence to signal transduction terminology as employed in the animal research field and in molecular biology textbooks, to simplify and correct current models about JA signal transduction leading to gene expression.

Source: Journal of Plant Growth Regulation (2005) Vol. 23, p. 200-210