Yoko Yasuno

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.

Structure and Mechanism of the Monoterpene Cyclolavandulyl Diphosphate Synthase that Catalyzes Consecutive Condensation and Cyclization

Here, we report the three-dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyses the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclisation to form a cyclic monoterpene, CLPP. The structures of apo-CLDS and CLDS in complex with Tris, pyrophosphate, and Mg ion were refined at 2.00 Å resolution and 1.73 Å resolution, respectively. CLDS adopts a typical fold for cis-prenyl synthases and forms a homo-dimeric structure. An in vitro reaction using a regiospecifically H-substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure-based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase-catalysed reactions. Terpenoids represent an important class of biologically active compounds owing to their structural diversity. The diverse structures of terpenoids are derived from two simple 5-carbon units: isopentenyl diphosphate (IPP) and DMAPP. An isoprenyl diphosphate synthase (IDS) catalyses the canonical and sequential “head-to-tail” condensation of IPP to DMAPP to produce a longer acyclic polyprenyl diphosphate such as C10 geranyl diphosphate (GPP) (eq. 1 in Scheme 1a). The resulting acyclic polyprenyl diphosphate substrates can be cyclized by terpene cyclases into single-ring or multi-ring products, which can be further diversified by subsequent biosynthetic modifications. Thus, in terpenoid biosynthesis, two independent enzymes, an IDS and a cyclase, consecutively catalyse the condensation and cyclisation reactions, respectively, yielding structurally diverse terpenoids. Intriguingly, however, CLDS alone catalyses the “head-to-middle” condensation of two molecules of DMAPP (1) as well as the subsequent cyclisation to form CLPP (2), which is a monoterpene with a branchedand cyclic-carbon skeleton (eq. 2 in Scheme 1a). Lavandulyl diphosphate synthase (LPPS) from Lavandula × intermedia also Scheme 1. Terpene synthase reactions. (a) eq. 1, condensation of IPP and DMAPP by GPP synthase; eq. 2, condensation of two molecules of DMAPP and subsequent cyclisation by CLDS; eq. 3, condensation of two molecules of DMAPP by LPPS; eq. 4, condensation of DMAPP and GPP by Mcl22. (b) Condensation of two molecules of the deuterium-substituted DMAPP substrate (4) and subsequent cyclisation catalysed by CLDS. To simplify the figure, only key deuterium atoms are shown in the intermediates, Int1, Int2 and Int3. [a] Dr. Takeo Tomita, Masaya Kobayashi, Prof. Dr. Makoto Nishiyama, Prof. Dr. Tomohisa Kuzuyama Biotechnology Research Centre, The University of Tokyo 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan E-mail: utkuz@mail.ecc.u-tokyo.ac.jp [b] Yuma Karita, Dr. Yoko Yasuno, Prof. Dr. Tetsuro Shinada Department of Material Science, Graduate School of Science, Osaka City University, Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan [+] These authors contributed equally to this work. Supporting information for this article is given via a link at the end of the document. OPP OPP

Upregulation of Juvenile Hormone Titers in Female Drosophila melanogaster Through Mating Experiences and Host Food Occupied by Eggs and Larvae

Juvenile hormone (JH) plays a crucial role in the determination of developmental timing in insects. In Drosophila melanogaster, reports indicate that JH titers are the highest immediately following eclosion and that the mating experience increases the titers in females. However, the titers have not been successively measured for an extended period of time after eclosion. This study reveals that JH titers are increased after eclosion in virgin females when supplied with food that is occupied by eggs and larvae as well as in mated females. With the presence of eggs and larvae, food induced the virgin females to lay unfertilized eggs. When combined with previous work indicating that females are attracted to such food where they prefer to lay eggs, these results suggest that flies can prepare themselves to lay eggs by changing the titers of JH under the presence of growing larvae, ensuring that the food is an appropriate place to oviposit.

An Aromatic Farnesyltransferase Functions in Biosynthesis of the Anti-HIV Meroterpenoid Daurichromenic Acid

A farnesyl diphosphate-preferring aromatic prenyltransferase plays a key role in the biosynthetic pathway of daurichromenic acid, an anti-HIV meroterpenoid, in Rhododendron dauricum. Rhododendron dauricum produces daurichromenic acid, an anti-HIV meroterpenoid, via oxidative cyclization of the farnesyl group of grifolic acid. The prenyltransferase (PT) that synthesizes grifolic acid is a farnesyltransferase in plant specialized metabolism. In this study, we demonstrated that the isoprenoid moiety of grifolic acid is derived from the 2-C-methyl-d-erythritol-4-phosphate pathway that takes place in plastids. We explored candidate sequences of plastid-localized PT homologs and identified a cDNA for this PT, RdPT1, which shares moderate sequence similarity with known aromatic PTs. RdPT1 is expressed exclusively in the glandular scales, where daurichromenic acid accumulates. In addition, the gene product was targeted to plastids in plant cells. The recombinant RdPT1 regiospecifically synthesized grifolic acid from orsellinic acid and farnesyl diphosphate, demonstrating that RdPT1 is the farnesyltransferase involved in daurichromenic acid biosynthesis. This enzyme strictly preferred orsellinic acid as a prenyl acceptor, whereas it had a relaxed specificity for prenyl donor structures, also accepting geranyl and geranylgeranyl diphosphates with modest efficiency to synthesize prenyl chain analogs of grifolic acid. Such a broad specificity is a unique catalytic feature of RdPT1 that is not shared among secondary metabolic aromatic PTs in plants. We discuss the unusual substrate preference of RdPT1 using a molecular modeling approach. The biochemical properties as well as the localization of RdPT1 suggest that this enzyme produces meroterpenoids in glandular scales cooperatively with previously identified daurichromenic acid synthase, probably for chemical defense on the surface of R. dauricum plants.