Evolutionary Relationship and Substrate Specificity of Arabidopsis Thaliana Fatty Acid Omega-Hydroxylase

Plant Science 170 (2006) 326–338 www.elsevier.com/locate/plantsci

Evolutionary relationship and substrate specificity of Arabidopsis thaliana fatty acid omega-hydroxylase Ire`ne Benveniste a, Takeshi Saito b, Yong Wang c, Sylvie Kandel a, Hongwei Huang b, Franck Pinot a, Rachel A. Kahn a, Jean-Pierre Salau¨n d, Miyuki Shimoji e,* a IBMP-CNRS UPR 2357, De´partement Re´sponse Me´taboliques a l’Environnement Biotique, 28 rue Goethe F-67083 Strasbourg Cedex, France b Accelrys Inc., 3-3-1 Nishi-Shinbashi, Minato-ku, Tokyo 105-0003, Japan c De´partment de Biologie Molećulaire Ve´ge´tale, Baˆtiment de Biologie, Universite´ de Lausanne, CH-1015 Lausanne, Switzerland d Biochime et Biologie Molećulaire Faculte´ de Me´decine de Brest CS93837-29238, Brest-Cedex 3, France e Institute of Environmental Medicine, Division of Biochemical Toxicology, Karolinska Institutet, Box 210, S-17177 Stockholm, Sweden Received 8 April 2005; accepted 30 August 2005 Available online 3 October 2005

This paper is dedicated to Prof. Francis Durst on occasion of his retirement.

Abstract On the basis of a phylogenic tree constructed using the available Arabidopsis thaliana genomic sequence, new fatty acid (FA) omega- hydroxylases were cloned and expressed in yeast. Several uncharacterized cytochrome P450s (CYP, P450), comprising a phylogenic sub-cluster (CYP86A, 94B, 94C, 96A, 704A, 97B and 711A) demonstrated FA omega-hydroxylase activities towards saturated FAs with medium chain length. While CYP96A4 showed the highest catalytic activity among the enzymes characterized in this study, other CYP96A subfamily members did not display any potent activity. In addition, CYP704A2 and CYP711A1, which are phylogenetically distant to both CYP86 and CYP94 with FA omega- hydroxylase activities, did not show any metabolic conversion of FAs. As we have studied omega-hydroxylation of FAs catalyzed by a series of CYP94 isoforms, active site models were produced to compare inactive CYP704A2 and CYP711A1 to active CYP94C1. The modeled structures revealed that the hydrophobicity in the heme binding site is very different between active and inactive isoforms. CYP94C1 contains highly hydrophobic residues while CYP704A2 and CYP711A1 do not. Our study provides evidence that substrate specificity is conserved in phylogenetically related isoforms and suggests specific residues in the active site pocket that play a key role in determining substrate specificity. # 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Plant cytochrome P450; Fatty acid omega-hydroxylase; Phylogenic tree; Active site

1. Introduction

Abbreviations: AOS, allene oxide synthase; AT, acyltransferase; BSTFA, FA hydroxylases are classified as in-chain, alpha, beta and bis(trimethylsilyl) trifluoroacetamide; DAGAT, diacylglycerol acyltransferase; omega-hydroxylases [1–3]. Among these, P450s catalyze in C12:0, lauric acid; C14:0, myristic acid; C16:0, palmitic acid; C18:1, oleic chain, alpha and omega hydroxylase activities. Omega- acid; CoA, coenzyme A; CYP, P450, cytochrome P450; FA, fatty acid; JA, jasmonic acid; FAME, fatty acid methyl ester; GC/MS, gas chromatography/ hydroxylated FAs are signaling molecules, known as (i) mass spectrometry; LPC, lysophosphatidylcholine, 1-palmitoyl-sn-glycero-3- mediators of plant defense reactions [4–6] and (ii) high affinity phosphocholine; 1jpz.pdb, Protein Data Bank code 1jpz; P450BM-3, CYP102; ligands of peroxisome proliferator activated receptors (PPARs) P450cam, CYP101A1; PPAR, peroxisome proliferator activated receptor; PCR, in mammals controlling not only the lipid level [7] but polymerase chain reaction; SRS, substrate recognition site; TLC, thin-layer also blood pressure [8] and cell differentiation, etc. [9].Long chromatography * Corresponding author. Tel.: +46 8 52487954; fax: +46 8 343849. chain FAs are hydroxylated at in-chain positions by oleate E-mail address: [email protected] (M. Shimoji). 12-hydroxylase in plants [10].

Fig. 1. Relationships of plant FA hydroxylases with hydroxylases from other kingdoms. At the DDBJ web site: http://hypernig.nig.ac.jp/homology/clustalw-e.shtml, the sequences were aligned with Clustal W (1.83) and the tree was calculated. The phylogenic tree was drawn with TreeViewPPC 1.5.3. Plant FA omega-hydroxylases (CYP86 and CYP94) are more closely related to FA hydroxylases from animals (rat CYP4A1, rat 4B1, human 4F2), fungi (yeast CYP52A4) and bacteria (CYP102) than to most of the other plant P450s. A subcluster is formed by plant CYP86A1, 86A2, 94A1, 94A2, 94A3, 94A5, 94B3, 94C1, 96A4 and 704A2, yeast CYP52A4, bacteria CYP102, rat CYP4A1, 4B1 and 4F1, plant CYP72A7, 97B3 and cyanobacteria CYP110A2. It should be noted that CYP86, 97 and 711 constitute separate clans since the 86 clan contains the unique conserved PERW sequence in the so-called ‘‘PERF’’ region (between K0helix and heme-binding region), while 97 and 711 clans contain the more conserved PERF consensus. CYP711 is located near human CYP5 (thromboxane synthase). We were interested in whether plant CYP96, 97, 704 and 711 are positioned near the animal CYP2 family (several are FA hydroxylases, e.g. CYP2E1 [75,76] and arachidonic acid epoxygenase, e.g. CYP2J2 [53]). Therefore, P450 sequences were chosen from both kingdoms for producing our phylogenic tree. Actually, the CYP2 family is not close to either CYP96A4, 704A2, 711A1, or plant FA omega-hydroxylase (CYP86A and CYP94A subfamily). For a general overview the reader is referred to Nelson’s website http:// drnelson.utmem.edu/CytochromeP450.html which contains a variety of detailed phylogenic trees.

In plants, FA omega-hydroxylation is essential for the identity between species as shown in Fig. 1. In mammals, synthesis of the cuticle and is probably involved in signaling the CYP4 family members catalyze the omega-hydroxylation [11–14]. FAs of C16 and C18 chain length, that are polymerized of saturated, unsaturated and oxygenated FAs [23–25]. for the cuticle [11], are potent substrates of FA omega- Members of the CYP52 family has been reported as efficient hydroxylase in plant P450s, e.g. CYP94A1 [13,15]. Plants fatty acid hydroxylases in yeast [26]. In order to understand initiate oxylipin synthesis from linolenic acid whereas animals plant functions of FA omega-hydroxylases, we have cloned and utilize arachidonic acid [16,17]. Interestingly, both organisms characterized several omega-hydroxylases of FAs from Vicia synthesize cyclopentenones as final products: jasmonic acid sativa, Arabidopsis thaliana and Nicotiana tabacum that (JA), is specifically synthesized in plants being the most formed the first members of the CYP94 and CYP86 families important signaling FA derivative [18]; and prostaglandins that [15,27–29]. These CYP86A and CYP94A subfamilies are oxygenated derivatives of C20 polyunsaturated FAs appeared more closely related to other species P450s with including arachidonic acid [17]. In mammals, PPAR alpha FA hydroxylase activity as mentioned above (CYP4, 52 and regulates FA-related genes involved in lipid homeostasis 102) than to other plant P450s (Fig. 1). [19,20] whereas the corresponding plant receptors have not In the present study, we hypothesized that plant P450s been identified yet. Thus, plants and mammals display diversity related to CYP86A and CYP94A evolutionarily, were FA and similarity concerning lipid signaling molecules. omega-hydroxylases. The systematic cDNA cloning of A. Saturated FAs with medium chain lengths, lauric acid thaliana CYP86, 94, 96, 704, 97 and 711 was guided by the (C12:0), myristic acid (C14:0), palmitic acid (C16:0), are plant phylogenic tree (Fig. 2), and the catalytic capacity hydroxylated at the omega-position by P450s in a broad range towards FAs was defined. The catalytic properties of P450s are of species [3,21,22]. These hydroxylases share sequence important because it should provide a firm basis for forth- Download English Version: https://daneshyari.com/en/article/2018630

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