Tanetoshi Koyama

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. We prepared a set of comprehensive metabolic pathway maps for Arabidopsis (Arabidopsis thaliana) and depicted these graphically in Scalable Vector Graphics format. Individual transcripts assigned to a reaction are represented symbolically together with the symbols of the reaction and metabolites on metabolic pathway maps. Using quantitative values for transcripts and/or metabolites submitted by the user as Comma Separated Value-formatted text through the Internet, the KaPPA-View server inserts colored symbols corresponding to a defined metabolic process at that site on the maps and returns them to the users browser. The server also provides information on transcripts and metabolites in pop-up windows. To demonstrate the process, we describe the dataset obtained for transgenic plants that overexpress the PAP1 gene encoding a MYB transcription factor on metabolic pathway maps. The presentation of data in this manner is useful for viewing metabolic data in a way that facilitates the discussion of gene function.

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. Although many studies on structure–function relationships of cis‐prenyltransferases have been reported, regulation mechanisms for the ultimate prenyl chain length have not yet been elucidated. We report here that the ultimate chain length of prenyl products can be controlled through structural manipulation of UPP synthase of M.u2003luteus B‐P 26, based on comparisons between structures of various cis‐prenyltransferases. Replacements of Ala72, Phe73, and Trp78, which are located in the proximity of the substrate binding site, with Leuu2003−u2003as in Z,E‐farnesyl diphosphate (C15) synthaseu2003−u2003resulted in shorter ultimate products with C20−35. Additional mutation of F223H resulted in even shorter products. On the other hand, insertion of charged residues originating from long‐chain cis‐prenyltransferases into helix‐3, which participates in constitution of the large hydrophobic cleft, resulted in lengthening of the ultimate product chain length, leading to C60−75. These results helped us understand reaction mechanisms of cis‐prenyltransferase including regulation of the ultimate prenyl chain‐length.

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 (CH3OGPP) as allylic substrate homologs. The wild-type FPP synthase reaction of HOGPP (and CH3OGPP) 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. Moreover, the latter gave a double-condensation product, methoxygeranylgeranyl diphosphate, as the main product and only a trace of methoxyfarnesyl diphosphate was obtained.

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. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.