Crystal structure of Thermobifida fusca cis-prenyltransferase reveals the dynamic nature of its RXG motif-mediated inter-subunit interactions critical for its catalytic activity.

cis-Prenyltransferases (cis-PTs) catalyze consecutive condensations of isopentenyl diphosphate to an allylic diphosphate acceptor to produce a linear polyprenyl diphosphate of designated length. Dimer formation is a prerequisite for cis-PTs to catalyze all cis-prenyl condensation reactions. The structure-function relationship of a conserved C-terminal RXG motif in cis-PTs that forms inter-subunit interactions and has a role in catalytic activity has attracted much attention.

Purification and characterization of small and large rubber particles from Hevea brasiliensis.

Natural rubber (NR) is synthesized by the rubber transferase (RTase) on rubber particles (RPs) in latex. Due to the heterogeneity of the RPs in latex, it is difficult to precisely characterize the RTase activity. In this study, we separated the RPs of Hevea brasiliensis with different particle size distributions, via stepwise centrifugations.

Identification of laticifer-specific genes and their promoter regions from a natural rubber producing plant Hevea brasiliensis.

Latex, the milky cytoplasm of highly differentiated cells called laticifers, from Hevea brasiliensis is a key source of commercial natural rubber production. One way to enhance natural rubber production would be to express genes involved in natural rubber biosynthesis by a laticifer-specific overexpression system. As a first step to identify promoters which could regulate the laticifer-specific expression, we identified random clones from a cDNA library of H.

Coordinated transcriptional regulation of isopentenyl diphosphate biosynthetic pathway enzymes in plastids by phytochrome-interacting factor 5.

All isoprenoids are derived from a common C5 unit, isopentenyl diphosphate (IPP). In plants, IPP is synthesized via two distinct pathways; the cytosolic mevalonate pathway and the plastidial non-mevalonate (MEP) pathway. In this study, we used a co-expression analysis to identify transcription factors that coordinately regulate the expression of multiple genes encoding enzymes in the IPP biosynthetic pathway.

Identification and characterization of a cis,trans-mixed heptaprenyl diphosphate synthase from Arabidopsis thaliana.

In eukaryotes, dolichols (C(70-120)) play indispensable roles as glycosyl carrier lipids in the biosynthesis of glycoproteins on endoplasmic reticulum. In addition to dolichols, seed plants have other types of Z,E-mixed polyisoprenoids termed ficaprenol (tri-trans,poly-cis-polyprenol, C(45-75)) and betulaprenol (di-trans,poly-cis-polyprenol, C(30-45) and C(≥70)) in abundance. However, the physiological significance of these polyprenols has not been elucidated because of limited information regarding cis-prenyltransferases (cPTs) which catalyze the formation of the structural backbone of Z,E-mixed polyisoprenoids.

Crystal structure of heterodimeric hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 reveals that the small subunit is directly involved in the product chain length regulation.

Hexaprenyl diphosphate synthase from Micrococcus luteus B-P 26 (Ml-HexPPs) is a heterooligomeric type trans-prenyltransferase catalyzing consecutive head-to-tail condensations of three molecules of isopentenyl diphosphates (C(5)) on a farnesyl diphosphate (FPP; C(15)) to form an (all-E) hexaprenyl diphosphate (HexPP; C(30)). Ml-HexPPs is known to function as a heterodimer of two different subunits, small and large subunits called HexA and HexB, respectively. Compared with homooligomeric trans-prenyltransferases, the molecular mechanism of heterooligomeric trans-prenyltransferases is not yet clearly understood, particularly with respect to the role of the small subunits lacking the catalytic motifs conserved in most known trans-prenyltransferases.

In vitro synthesis of high molecular weight rubber by Hevea small rubber particles.

Hevea brasiliensis is one of few higher plants producing the commercial natural rubber used in many significant applications. The biosynthesis of high molecular weight rubber molecules by the higher plants has not been clarified yet. Here, the in vitro rubber biosynthesis was performed by using enzymatically active small rubber particles (SRP) from Hevea.

Cloning and functional analysis of cis-prenyltransferase from Thermobifida fusca.

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by a condensation of isopentenyl diphosphate to an allylic diphosphate. A novel gene encoding a cis-prenyltransferase is cloned from Thermobifida fusca. It showed a unique substrate specificity accepting dimethylallyl diphosphate as a shortest allylic substrate, and synthesizes polyprenyl products up to C(70).

Product chain-length determination mechanism of Z,E-farnesyl diphosphate synthase.

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z,E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z,E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z,E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086.

Cloning and functional analysis of novel short-chain cis-prenyltransferases.

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by sequential condensation of isopentenyl diphosphate with allylic diphosphate. cis-Prenyltransferases can be classified into three subgroups: short-, medium-, and long-chain cis-prenyltransferase, according to their product chain lengths. cis-Farnesyl diphosphate synthase from Mycobacterium tuberculosis has been the only example as short-chain cis-prenyltransferase so far characterized.

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction.

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions.

Kinetic studies of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase reaction using 3-desmethyl allylic substrate analogs.

In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate.

Exploring biosynthetic diversity with trichodiene synthase.

Trichodiene synthase is a terpenoid cyclase that catalyzes the cyclization of farnesyl diphosphate (FPP) to form the bicyclic sesquiterpene hydrocarbon trichodiene (89%), at least five sesquiterpene side products (11%), and inorganic pyrophosphate (PP(i)). Incubation of trichodiene synthase with 2-fluorofarnesyl diphosphate or 4-methylfarnesyl diphosphate similarly yields sesquiterpene mixtures despite the electronic effects or steric bulk introduced by substrate derivatization. The versatility of the enzyme is also demonstrated in the 2.

Substrate specificities of wild and mutated farnesyl diphosphate synthases from Bacillus stearothermophilus with artificial substrates.

To determine the substrate specificities of wild and mutated types of farnesyl diphosphate (FPP) synthases from Bacillus stearothermophilus, we examined the reactivities of 8-hydroxygeranyl diphosphate (HOGPP) and 8-methoxygeranyl diphosphate (CH(3)OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH(3)OGPP) with isopentenyl diphosphate (IPP) gave hydroxyfarnesyl- (and methoxyfarnesyl-) diphosphates that stopped at the first stage of condensation. On the other hand, with mutated type FPP synthase (Y81S), the former gave hydroxygeranylgeranyl diphosphate as the main double-condensation product together with hydroxyfarnesyl diphosphate as a single-condensation product and a small amount of hydroxygeranylfarnesyl diphosphate as a triple-condensation product.

Novel fluorescent analogues for transmembrane movement study of polyprenyl phosphates.

In order to investigate the transmembrane movement of polyprenyl phosphate across biological membranes, NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl)-labeled polyprenyl phosphate analogues were prepared. These analogues proved to be possible tools for a direct observation of the transmembrane flip-flop movement of polyprenyl phosphates by use of a sodium dithionite-quenching procedure.

Direct observation of substrate-enzyme complexation by surface forces measurement.

The substrate-enzyme complexation of heptaprenyl diphosphate synthase was directly investigated using colloidal probe atomic force microscopy (AFM) and a quartz crystal microbalance (QCM) in order to obtain new insights into the molecular mechanism of the enzyme reaction. This enzyme is composed of two dissociable subunits that exhibit a catalytic activity only when they are associated together in the presence of a cofactor, Mg2+, and a substrate, farnesyl diphosphate (FPP). The QCM measurement revealed that FPP was preferentially bound to subunit II in the presence of Mg2+, while the AFM measurement showed that the adhesive force between the subunits was observed only in the presence of both Mg2+ and FPP.

Structure and function of cis-prenyl chain elongating enzymes.

All carbon skeletons of isoprenoids, whose chain lengths vary widely from geranyl diphosphate (C10) to natural rubber (C>10,000), are synthesized by sequential condensation of isopentenyl diphosphate with an allylic diphosphate through catalytic functions of a group of enzymes commonly called "prenyltransferases." Prenyltransferases are classified into two major groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases, according to the geometry of the prenyl chain units in the products. From the year 1987, many genes encoding trans-prenyltransferases were cloned and clearly characterized.

Manipulation of prenyl chain length determination mechanism of cis-prenyltransferases.

The carbon backbones of Z,E-mixed isoprenoids are synthesized by sequential cis-condensation of isopentenyl diphosphate (IPP) and an allylic diphosphate through actions of a series of enzymes called cis-prenyltransferases. Recent molecular analyses of Micrococcus luteus B-P 26 undecaprenyl diphosphate (UPP, C55) synthase [Fujihashi M, Zhang Y-W, Higuchi Y, Li X-Y, Koyama T & Miki K (2001) Proc Natl Acad Sci USA98, 4337-4342.] showed that not only the primary structure but also the crystal structure of cis-prenyltransferases were totally different from those of trans-prenyltransferases.

KaPPA-view: a web-based analysis tool for integration of transcript and metabolite data on plant metabolic pathway maps.

The application of DNA array technology and chromatographic separation techniques coupled with mass spectrometry to transcriptomic and metabolomic analyses in plants has resulted in the generation of considerable quantitative data related to transcription and metabolism. The integration of "omic" data is one of the major concerns associated with research into identifying gene function. Thus, we developed a Web-based tool, KaPPA-View, for representing quantitative data for individual transcripts and/or metabolites on plant metabolic pathway maps.

Substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate.

In order to develop synthetic methods for biologically active homoallylic terpene sulfates, we examined the applicability and substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate (homoIPP). The reaction of dimethylallyl diphosphate with homoIPP by use of Bacillus stearothermophilus (all-trans)-farnesyl diphosphate synthase resulted in efficient yields of cis-(yield: 45.9%) and trans-4,8-dimethylnona-3,7-dien-1-ol (homoGOH, 25.

Involvement of Hevea latex organelle membrane proteins in the rubber biosynthesis activity and regulatory function.

Centrifugation of fresh Hevea rubber latex yields three distinct fractions. The sediment bottom fraction (BF) content of membrane-bound organelles is ca. 20 vol.

In vivo interaction between the human dehydrodolichyl diphosphate synthase and the Niemann-Pick C2 protein revealed by a yeast two-hybrid system.

Dehydrodolichyl diphosphate (DedolPP) synthase catalyzes the sequential condensation of isopentenyl diphosphate with farnesyl diphosphate to synthesize DedolPP, a biosynthetic precursor for dolichol which plays an important role as a sugar-carrier lipid in the biosynthesis of glycoprotein in eukaryotic cells. During certain pathological processes like Alzheimer's disease or some neurological disorders, dolichol has been shown to accumulate in human brain. In order to understand the regulatory mechanism of dolichol in eukaryotes, we performed a yeast two-hybrid screen using full length human DedolPP synthase gene [Endo et al.

Significance of highly conserved aromatic residues in Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

Undecaprenyl diphosphate synthase catalyzes the sequential condensation of eight molecules of isopentenyl diphosphate (IPP) in the cis-configuration into farnesyl diphosphate (FPP) to produce undecaprenyl diphosphate (UPP), which is indispensable for the biosynthesis of the bacterial cell wall. This cis-type prenyltransferase exhibits a quite different mode of binding of homoallylic substrate IPP from that of trans-type prenyltransferase [Kharel Y. et al.

Molecular cloning, expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis. A key factor participating in natural rubber biosynthesis.

Natural rubber from Hevea brasiliensis is a high molecular mass polymer of isoprene units with cis-configuration. The enzyme responsible for the cis-1,4-polymerization of isoprene units has been idengified as a particle-bound rubber transferase, but no gene encoding this enzyme has been cloned from rubber-producing plants. By using sequence information from the conserved regions of cis-prenyl chain elongating enzymes that were cloned recently, we have isolated and characterized cDNAs from H.

Mutational analysis of allylic substrate binding site of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase.

Undecaprenyl diphosphate (UPP) synthase catalyzes the sequential cis-condensation of isopentenyl diphosphate (IPP) onto (E,E)-farnesyl diphosphate (FPP). In our previous reports on the Micrococcus luteus B-P 26 UPP synthase, we have shown that the conserved residues in the disordered region from Ser-74 to Val-85 is crucial for the binding of FPP and the catalytic function [Fujikura, K., et al.