Separation of enzymatic functions and variation of spin state of rice allene oxide synthase-1 by mutation of Phe-92 and Pro-430
Graphical abstract
Introduction
The biosynthesis of lipid signaling molecules is usually initiated by dioxygenation of polyunsaturated fatty acids (PUFA) by lipoxygenase (LOX) and the resulting hydroperoxy fatty acids are subsequently metabolized by cytochrome P450 enzymes. In animals, the eicosanoid pathway converts arachidonic acid (C20) to prostaglandins or thromboxane, which play important roles in vascular hemostasis [1]. In plants, the octadecanoid pathway is the biosynthetic pathway for oxylipins, which play critical roles in the response to biotic and abiotic stress [2]. Biosynthesis of oxylipins which involves several branching pathways [2], [3], [4], [5], [6], initiates with the oxygenation of linoleic acid (LA) or linolenic acid (LnA) at the C-9 or C-13 positions by lipoxygenase (LOX) [7]. LOX generates 9- and 13-hydroperoxy fatty acids, which are then metabolized by three closely-related members of the ubiquitous cytochrome P450 subfamily 74 (CYP74): allene oxide synthase (AOS), hydroperoxide lyase (HPL), and divinyl ether synthase (DES) [3], [5], [8]. CYP74 family enzymes do not require molecular oxygen or redox partners, because they are able to utilize the activated hydroperoxo moiety of the substrate [9], [10]. In plants, AOS and HPL are required for biosynthesis of jasmonates and aldehydes, respectively, which are involved in resistance to herbivores and disease and the general stress response. While many studies focus on the importance of AOS and jasmonates for plant defense mechanisms, the contribution of HPL and aldehydes are less well characterized. Several studies suggest that the two signaling cascades are interdependent, interrelated, and engaged in cross-talk. Thus, both branches of the oxylipin biosynthetic pathway may play critical roles in plant defense signaling [11], [12]. The 3-dimensional structure of AOS has been inferred from X-ray crystal structures of Arabidopsis thaliana AOS (AtAOS) [13] and Parthenium argentatum AOS (PaAOS) [14]. AOS exhibits a classical cytochrome P450 fold, with a long binding loop and unique I-helix [13], [14], which are shared structural motifs of all CYP74 enzymes. The significance of species-specific variations in catalytic residues of AOS enzymes from dicot plants, but not monocot plants, has been examined by site-directed mutagenesis. For example, Phe-137 in AtAOS [13], Phe-295 and Ser-297 in tomato AOS [9], and Glu-292 and Val-379 in Nicotiana DES [10] were mutated and shown to play important roles in enzyme catalysis.
The dual positional substrate specific rice allene oxide synthase-1 (OsAOS1) localizes to the chloroplast and its solubility and oligomeric state is strongly dependent on detergents [15]. The OsAOS1 reaction product, allene oxide, is converted to the α-ketol or γ-ketol derivative by nonenzymatic hydration, as observed previously for other AOS enzymes (Fig. 1). Type II ligands inhibit the catalytic activity of OsAOS1 with concomitant conversion of iron spin state from high to low [16], [17]. The goal of the current study was to identify amino acid residues in OsAOS1 that are specifically required for one of the two branches of the catalytic pathway, the AOS or the HPL branch, and/or to identify amino acid residues that regulate the heme iron spin state transition of OsAOS1. Enzymes carrying mutations in these residues, if identified, would preferentially facilitate the AOS or the HPL branch of the pathway, and demonstrate that the two catalytic mechanisms map to distinct domains and/or residues of the OsAOS1 polypeptide. Here, the cloned OsAOS1 coding sequence was altered by site-directed mutagenesis and several OsAOS1 enzyme variants were characterized with respect to UV–vis spectra, spin state distribution, sensitivity to inhibition by imidazole, and relative partitioning of substrate to the AOS and HPL branches of the catalytic pathway. Implications of the results for understanding the biological roles of CYP74 family proteins and plant defense mechanisms are discussed.
Section snippets
Bioinformatics and site-directed mutagenesis
Amino acid sequences of CYP74 family members were obtained by searching NCBI and were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). The three-dimensional structure of OsAOS1 was modeled using automated mode on the SWISS-MODEL server (http://swissmodel.expasy.org). Graphic output was generated with Pymol [18]. Mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene, USA) and pET-28b-derived constructs into which OsAOS1 coding sequence was
Strategy for site-directed mutation
Previous studies revealed that Phe-137 of AtAOS stabilizes the positively-charged carbocation intermediate through a cation-π interaction, which is essential for the AOS reaction. In HPL and DES, the equivalent Phe residue is replaced by leucine (Fig. 2A). Consistent with this, mutation of Phe-137 in AtAOS to leucine increased the HPL activity of the enzyme [13]. Asn-278 and Cys-429 of OsAOS1 are conserved in all CYP74 family enzymes. Asn-278 binds to the hydroperoxo group of substrate, and
Discussion
OsAOS1 exhibits dual positional substrate specificity, showing a strong preference for (13S)-HPOT, which is a committed precursor for biosynthesis of JA from linolenic acid via the 13-oxylipin biosynthetic pathway [16]. However, OsAOS1 also plays a role in synthesis of aldehydes from linoleic and linolenic acid via its HPL activity. The AOS activity of OsAOS1 is strongly inhibited by imidazole and its derivatives, well known competitive inhibitors [21], [22] and type II ligands of AOS that
Acknowledgments
This work was supported by a National Research Foundation grant (2011-0008591) to O. Han.
References (24)
- et al.
J. Lipid. Mediat. Cell Signal.
(1995) - et al.
Prog. Lipid Res.
(2009) Prog. Lipid Res.
(1998)- et al.
Curr. Opin. Plant Biol.
(2002) - et al.
FEBS Lett.
(2008) - et al.
FEBS Lett.
(2013) Curr. Opin. Plant Biol.
(2006)- et al.
Bioorg. Med. Chem.
(2002) - et al.
FEBS Lett.
(2006) - et al.
PLoS ONE
(2012)