Substrate Specificities of <i>E</i>-and <i>Z</i>-Farnesyl Diphosphate Synthases with Artificial Substrates

Abstract

Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan *Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Kojirakawa-machi, Yamagata 990-8560, Japan **Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan Fax: 81-172-39-3947, e-mail: nagaki@cc.hirosaki-u.ac.jp Here, to assess substrate specificities of E-and Z-farnesyl diphosphate synthases from Bacillus stearothermophilus and Thermobifida fusca, we examined the reactivities of isopentenyl diphosphate homologs having an alkyl group at the 3-position. E- and Z-FPP synthase reactions of geranyl diphosphate (GPP) with 3-ethylisopentenyl diphosphate produced E- and Z-3-ethylfarnesyl diphosphates (yield: 70.8%, and 11.3%, respectively). The E-FPP synthase reaction of GPP with 3-propylisopentenyl diphosphate produced E-3-propylfarnesyl diphosphate (yield: 40.1%); however, when reacting GPP with 3-propylisopentenyl diphosphate by Z-FPP synthase reaction, only trace product was acquired. Further, neither E- nor Z-FPP synthase reaction of GPP with 3-butylisopentanyl diphosphate produced any product. Key words: E- and Z-farnesyl diphosphate synthases, substrate specificity, homologs of isopentenyl diphosphate, Bacillus stearothermophilus, Thermobifida fusca 1. INTRODUCTION Prenyltransferases can be roughly divided into Z- and E-prenyl chain elongating enzymes[1-5]. E-farnesyl diphosphate (FPP) synthase, a short prenyl chain elongating enzyme, has long been the subject of intense research. The E-FPP synthase has been solved to quite detailed portions, such as a chain length determination mechanism [6-8]. However, after Crick et al. successfully cloned Z-FPP synthase from Mycobacterium tubercule in 2000, a great number of studies began focusing on Z-prenyltransferase[9-11]. Subsequent to Crick et al.’s findings, Koyama et al. recently successfully cloned Z-FPP synthase from several other bacterial species, including Thermobifida fusca and Corynebacterium glutamicum [12-14]. C. Sallaud et al. identified the genes encoding Z-FPP synthase in the wild tomato, Solanum habrochaites, and reported that the recombinant enzymatic reaction between dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) produced all-Z-FPP [15]. E-FPP synthase [EC 2.5.1.10] catalyzes the head-to-tail condensation of IPP with DMAPP (or geranyl diphosphate [GPP]) as an allylic substrate, producing FPP as the final product (Scheme 1). Several previous studies have investigated the substrate specificities of FPP synthase with both allylic and homoallylic substrate homologs [16-20]. Scheme 1. FPP Synthase Reaction of DMAPP withIPP Our team also recently reported on the reactivities of several allylic substrate homologs which have a hydrophilic group at the ω-position, using B. stearothermophilus and porcine liver FPP synthases [21-23]. Previously, to examine the substrate specificity of FPP synthase derived from B. stearothermophilus, we reported on the reaction of GPP with IPP homologs which have an alkyl group at the 3-position[24]. Here, we compared E- and Z-FPP synthases by examining several new, similar reactions. We then assessed the substrate specificities of the synthases by examining the reactivity of artificial substrate homologs.