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Tokuzo Nishino - One of the best experts on this subject based on the ideXlab platform.
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an alternative mechanism of product chain length determination in type iii geranylgeranyl diphosphate synthase
FEBS Journal, 2003Co-Authors: Motoyoshi Noike, Tokuzo NishinoAbstract:(All-E) prenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl Diphosphates with allylic prenyl Diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl diphosphate synthases, i.e. farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III geranylgeranyl diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl diphosphate synthases.
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substrate specificity of thermostable farnesyl diphosphate synthase with respect to 4 alkyl group homologs of isopentenyl diphosphate
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Masahiko Nagaki, Junji Ishibashi, Yuji Maki, Tokuzo Nishino, Hiroto Yamamoto, Ayumi Takahashi, Tanetoshi KoyamaAbstract:In order to investigate substrate spcificity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPS), we examined the reactivity of 4-alkyl group homologs of isopentenyl diphosphate (IPP). The enzymatic reactions of the 4-methyl homologs, (E)-3-methylpent-3-enyl diphophates (1a) and (Z)-3-methylpent-3-enyl diphophates (1b) with geranyl diphosphate (GPP) gave 4-methylfarnesyl Diphosphates (2a and 2b), respectively. The stereochemistry of each aldehyde derived from 2a or 2b was determined by CD spectrometry to be (S)-4-methylfarnesal or (R)-4-methylfarnesal, respectively. Similarly, 1a reacted with dimethylallyl diphosphate (DMAPP) to give a mixture of (4S)-4-methylgeranyl Diphosphates (3a) and (4S,8S)-4,8-dimethylfarnesyl Diphosphates (4a). The (Z)-isomer 1b also reacted with DMAPP to give the corresponding enantiomers with (4R)- and (4R,8R)-configurations. On the other hand, reactions of the 4-ethyl homologs, (E)-3-methylhex-3-enyl Diphosphates (1c) and (Z)-3-methylhex-3-enyl Diphosphates (1d) with GPP gave two types of 4-ethylfarnesyl Diphosphates. Reactions of 1c or 1d with DMAPP also gave two types of 4-ethylgeranyl- and 4,8-diethylfarnesyl Diphosphates. Meanwhile, reaction of the 4-propyl homologs, (E)-3-methylhept-3-enyl Diphosphates (1e) and (Z)-3-methylhept-3-enyl Diphosphates (1f) with GPP gave two types of 4-propylfarnesyl Diphosphates. Reactions of 1e or 1f with DMAPP gave only two types of the 4-propyl GPPs. However, neither (E)-3-methyloct-3-enyl Diphosphates (1g) or (Z)-3-methyloct-3-enyl Diphosphates (1h), nor (E)-4-bromo-3-methylbut-3-enyl diphosphate (1i) or (Z)-4-bromo-3-methylbut-3-enyl diphosphate (1j) was acceptable as a substrate for the thermophilic FPS at all.
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artificial substrates for undecaprenyl diphosphate synthase from micrococcus luteus b p 26
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Masahiko Nagaki, Yuji Maki, Tokuzo Nishino, Shunsuke Sato, Tanetoshi KoyamaAbstract:Abstract Substrate specificity of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 was investigated with respect to some alkyl- and bromo-group homologs of isopentenyl diphosphate. Among the homologs relating to the 3-methyl group, but-3-enyl diphosphate ( 2b ) and 3-ethylbut-3-enyl diphosphate ( 3b ) were accepted as substrates, with (all- E )-farnesyl diphosphate (FPP) to give 7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate, and a mixture of 3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and 3,7-diethyleicosa-2,6,10,14,18-pentaenyl Diphosphates, respectively. With respect to the homologs modified at the 4 position of isopentenyl diphosphate, (4 E )-3-methylpent-3-enyl diphosphate ( 2f ) was accepted as a substrate to give (4 S )-(2 Z ,6 E ,10 E ,14 E )-4-methylgeranylgeranyl- and (4 S ,8 S )-(2 Z ,6 Z ,10 E ,14 E ,18 E )-4,8-dimethylgeranylfarnesyl Diphosphates. Neither (4 Z )-3-methylpent-3-enyl diphosphate nor 4-bromo-3-methylbut-3-enyl Diphosphates was accepted as a substrate at all.
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a pathway where polyprenyl diphosphate elongates in prenyltransferase insight into a common mechanism of chain length determination of prenyltransferases
Journal of Biological Chemistry, 1998Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Naoki Tsuruoka, Masayasu Yano, Hiroyuki Nakane, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl Diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an α-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996)J. Biol. Chem. 271, 18831–18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.
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conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase two amino acids before the first aspartate rich motif solely determine eukaryotic farnesyl diphosphate synthase activity
Journal of Biological Chemistry, 1997Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Tokuzo NishinoAbstract:Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic Diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.
Shin-ichi Ohnuma - One of the best experts on this subject based on the ideXlab platform.
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a pathway where polyprenyl diphosphate elongates in prenyltransferase insight into a common mechanism of chain length determination of prenyltransferases
Journal of Biological Chemistry, 1998Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Naoki Tsuruoka, Masayasu Yano, Hiroyuki Nakane, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl Diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an α-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996)J. Biol. Chem. 271, 18831–18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.
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conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase two amino acids before the first aspartate rich motif solely determine eukaryotic farnesyl diphosphate synthase activity
Journal of Biological Chemistry, 1997Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Tokuzo NishinoAbstract:Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic Diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.
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a role of the amino acid residue located on the fifth position before the first aspartate rich motif of farnesyl diphosphate synthase on determination of the final product
Journal of Biological Chemistry, 1996Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Chika Ishida, Takeshi Nakazawa, Keishi Narita, Yoshie Takeuchi, Tokuzo NishinoAbstract:Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl Diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.
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Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION
The Journal of biological chemistry, 1996Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Chika Ishida, Kazutake Hirooka, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic Diphosphates to produce prenyl Diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from approximately 1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl Diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T.(1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.
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conversion from farnesyl diphosphate synthase to geranylgeranyl diphosphate synthase by random chemical mutagenesis
Journal of Biological Chemistry, 1996Co-Authors: Shin-ichi Ohnuma, Kyozo Ogura, Tanetoshi Koyama, Hisashi Hemmi, Takeshi Nakazawa, Annamaria Hallberg, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic Diphosphates to produce prenyl Diphosphates whose chain lengths are absolutely determined by each enzyme. In order to investigate the mechanisms of the consecutive reaction and of the determination of ultimate chain length, a random mutational approach was planned. The farnesyl diphosphate (FPP) synthase gene of Bacillus stearothermophilus was subjected to random mutagenesis by NaNO2 treatment to construct libraries of mutated FPP synthase genes on a high-copy plasmid. From the libraries, the mutants that showed the activity of geranylgeranyl diphosphate (GGPP) synthase were selected by the red-white screening method (Ohnuma, S.-i., Suzuki, M., and Nishino, T. (1994) J. Biol. Chem. 268, 14792-14797), which utilized carotenoid synthetic genes, phytoene synthase, and phytoene desaturase, to visualize the formation of GGPP in vivo. Eleven red positive clones were identified from about 24,300 mutants, and four (mutant 1, 2, 3, and 4) of them were analyzed for the enzyme activities. Results of in vitro assays demonstrated that all these mutants produced (all-E)-GGPP although the amounts were different. Each mutant was found to contain a few amino acid substitutions: mutant 1, Y81H and L275S; mutant 2, L34V and R59Q; mutant 3, V157A and H182Y; mutant 4, Y81H, P239R, and A265T. Site-directed mutagenesis showed that Y81H, L34V, or V157A was essential for the expression of the activity of GGPP synthase. Especially, the replacement of tyrosine 81 by histidine is the most effective because the production ratios of GGPP to FPP in mutant 1 and 4 are the largest. Based on prediction of the secondary structure, it is revealed that the tyrosine 81 situates on a point 11 approximately 12 A apart from the first DDXXD motif, whose distance is similar to the length of hydrocarbon moiety of FPP. These data might suggest that the aromatic ring of tyrosine 81 blocks the chain elongation longer than FPP. Comparisons of kinetic parameters of the mutated and wild type enzymes revealed several phenomena that may relate with the change of the ultimate chain length. They are a decrease of the total reaction rate, increase of Kmfor dimethylallyl diphosphate, decrease of Vmax for dimethylallyl diphosphate, and allylic substrate dependence of Km for IPP.
Tanetoshi Koyama - One of the best experts on this subject based on the ideXlab platform.
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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
Journal of Biological Chemistry, 2011Co-Authors: Daisuke Sasaki, Motoyoshi Noike, Tanetoshi Koyama, M Fujihashi, Naomi Okuyama, Y Kobayashi, Kunio MikiAbstract: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 (C5) on a farnesyl diphosphate (FPP; C15) to form an (all-E) hexaprenyl diphosphate (HexPP; C30). 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. We have determined the crystal structure of Ml-HexPPs both in the substrate-free form and in complex with 7,11-dimethyl-2,6,10-dodecatrien-1-yl diphosphate ammonium salt (3-DesMe-FPP), an analog of FPP. The structure of HexB is composed of mostly antiparallel α-helices joined by connecting loops. Two aspartate-rich motifs (designated the first and second aspartate-rich motifs) and the other characteristic motifs in HexB are located around the diphosphate part of 3-DesMe-FPP. Despite the very low amino acid sequence identity and the distinct polypeptide chain lengths between HexA and HexB, the structure of HexA is quite similar to that of HexB. The aliphatic tail of 3-DesMe-FPP is accommodated in a large hydrophobic cleft starting from HexB and penetrating to the inside of HexA. These structural features suggest that HexB catalyzes the condensation reactions and that HexA is directly involved in the product chain length control in cooperation with HexB.
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cloning expression and characterization of a functional cdna clone encoding geranylgeranyl diphosphate synthase of hevea brasiliensis
Biochimica et Biophysica Acta, 2003Co-Authors: Akiyuki Takaya, Yuanwei Zhang, Dhirayos Wititsuwannakul, Kasem Asawatreratanakul, Rapepun Wititsuwannakul, Seiji Takahashi, Tanetoshi KoyamaAbstract:Abstract Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic Diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent Km values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl Diphosphates of the GGPP synthase purified with Ni2+-affinity column were 24.1, 6.8, 2.3, and 11.5 μM, respectively. The enzyme shows optimum activity at approximately 40 °C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.
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substrate specificity of thermostable farnesyl diphosphate synthase with respect to 4 alkyl group homologs of isopentenyl diphosphate
Journal of Molecular Catalysis B-enzymatic, 2002Co-Authors: Masahiko Nagaki, Junji Ishibashi, Yuji Maki, Tokuzo Nishino, Hiroto Yamamoto, Ayumi Takahashi, Tanetoshi KoyamaAbstract:In order to investigate substrate spcificity of Bacillus stearothermophilus farnesyl diphosphate synthase (FPS), we examined the reactivity of 4-alkyl group homologs of isopentenyl diphosphate (IPP). The enzymatic reactions of the 4-methyl homologs, (E)-3-methylpent-3-enyl diphophates (1a) and (Z)-3-methylpent-3-enyl diphophates (1b) with geranyl diphosphate (GPP) gave 4-methylfarnesyl Diphosphates (2a and 2b), respectively. The stereochemistry of each aldehyde derived from 2a or 2b was determined by CD spectrometry to be (S)-4-methylfarnesal or (R)-4-methylfarnesal, respectively. Similarly, 1a reacted with dimethylallyl diphosphate (DMAPP) to give a mixture of (4S)-4-methylgeranyl Diphosphates (3a) and (4S,8S)-4,8-dimethylfarnesyl Diphosphates (4a). The (Z)-isomer 1b also reacted with DMAPP to give the corresponding enantiomers with (4R)- and (4R,8R)-configurations. On the other hand, reactions of the 4-ethyl homologs, (E)-3-methylhex-3-enyl Diphosphates (1c) and (Z)-3-methylhex-3-enyl Diphosphates (1d) with GPP gave two types of 4-ethylfarnesyl Diphosphates. Reactions of 1c or 1d with DMAPP also gave two types of 4-ethylgeranyl- and 4,8-diethylfarnesyl Diphosphates. Meanwhile, reaction of the 4-propyl homologs, (E)-3-methylhept-3-enyl Diphosphates (1e) and (Z)-3-methylhept-3-enyl Diphosphates (1f) with GPP gave two types of 4-propylfarnesyl Diphosphates. Reactions of 1e or 1f with DMAPP gave only two types of the 4-propyl GPPs. However, neither (E)-3-methyloct-3-enyl Diphosphates (1g) or (Z)-3-methyloct-3-enyl Diphosphates (1h), nor (E)-4-bromo-3-methylbut-3-enyl diphosphate (1i) or (Z)-4-bromo-3-methylbut-3-enyl diphosphate (1j) was acceptable as a substrate for the thermophilic FPS at all.
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identification of significant residues for homoallylic substrate binding of micrococcus luteus b p 26 undecaprenyl diphosphate synthase
Journal of Biological Chemistry, 2001Co-Authors: Yugesh Kharel, Kunio Miki, M Fujihashi, Yuanwei Zhang, Tanetoshi KoyamaAbstract:The primary structure of cis-prenyltransferase is totally different from those of trans-prenyltransferases (Shimizu, N., Koyama, T., and Ogura, K. (1998) J. Biol. Chem. 272, 19476-19481). To better understand the molecular mechanism of enzymatic cis-prenyl chain elongation, we selected seven charged residues in the conserved Region V and two of Phe-Ser motif in Region III of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 for substitutions by site-directed mutagenesis and examined their effects on substrate binding and catalysis. Kinetic studies indicated that replacements of Arg-197 or Arg-203 with Ser, and Glu-216 with Gln resulted in 7-11-fold increases of Km values for isopentenyl diphosphate and 18-1200-fold decreases of kcat values compared with those of the wild-type enzyme. In addition, two mutants with respect to the Phe-Ser motif in Region III, F73A and S74A, showed 16-32-fold larger Km values for isopentenyl diphosphate and 12-16-fold lower kcat values than those of the wild-type. Furthermore, product analysis indicated that three mutants, F73A, S74A, and E216Q, yielded shorter chain prenyl Diphosphates as their main products. These facts together with the protein structural analysis recently carried out (Fujihashi, M., Zhang, Y.-W., Higuchi, Y., Li, X.-Y., Koyama, T., and Miki, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4337-4342) indicated that the diphosphate moiety of homoallylic substrate is electrostatically recognized by the three charged amino acids, Arg-197, Arg-203, and Glu-216, in Region V and the Phe-Ser motif in Region III, also indispensable for homoallylic substrate binding as well as catalytic function. It was suggested that the undecaprenyl diphosphate synthase takes a different mode for the binding of isopentenyl diphosphate from that of trans-prenyl chain elongating enzymes.
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artificial substrates for undecaprenyl diphosphate synthase from micrococcus luteus b p 26
Journal of Molecular Catalysis B-enzymatic, 2000Co-Authors: Masahiko Nagaki, Yuji Maki, Tokuzo Nishino, Shunsuke Sato, Tanetoshi KoyamaAbstract:Abstract Substrate specificity of undecaprenyl diphosphate synthase of Micrococcus luteus B-P 26 was investigated with respect to some alkyl- and bromo-group homologs of isopentenyl diphosphate. Among the homologs relating to the 3-methyl group, but-3-enyl diphosphate ( 2b ) and 3-ethylbut-3-enyl diphosphate ( 3b ) were accepted as substrates, with (all- E )-farnesyl diphosphate (FPP) to give 7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl diphosphate, and a mixture of 3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and 3,7-diethyleicosa-2,6,10,14,18-pentaenyl Diphosphates, respectively. With respect to the homologs modified at the 4 position of isopentenyl diphosphate, (4 E )-3-methylpent-3-enyl diphosphate ( 2f ) was accepted as a substrate to give (4 S )-(2 Z ,6 E ,10 E ,14 E )-4-methylgeranylgeranyl- and (4 S ,8 S )-(2 Z ,6 Z ,10 E ,14 E ,18 E )-4,8-dimethylgeranylfarnesyl Diphosphates. Neither (4 Z )-3-methylpent-3-enyl diphosphate nor 4-bromo-3-methylbut-3-enyl Diphosphates was accepted as a substrate at all.
Chikara Ohto - One of the best experts on this subject based on the ideXlab platform.
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a pathway where polyprenyl diphosphate elongates in prenyltransferase insight into a common mechanism of chain length determination of prenyltransferases
Journal of Biological Chemistry, 1998Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Naoki Tsuruoka, Masayasu Yano, Hiroyuki Nakane, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensations of isopentenyl diphosphate to produce linear polyprenyl Diphosphates. Each enzyme forms the final product with a specific chain length. The product specificity of an enzyme is thought to be determined by the structure around the unknown path through which the product elongates in the enzyme. To explore the path, we introduced a few mutations at the 5th, the 8th, and/or the 11th positions before the first aspartate-rich motif of geranylgeranyl-diphosphate synthase or farnesyl-diphosphate synthase. The side chains of these amino acids are situated on the same side of an α-helix. In geranylgeranyl-diphosphate synthase, a single mutated enzyme (F77S) mainly produces a C25 product (Ohnuma, S.-I., Hirooka, K., Hemmi, H., Ishida, C., Ohto, C., and Nishino, T. (1996)J. Biol. Chem. 271, 18831–18837). A double mutated enzyme (L74G and F77G) mainly produces a C35 compound with significant amounts of C30 and C40. A triple mutated enzyme (I71G, L74G, and F77G) mainly produces a C40compound with C35 and C45. Mutated farnesyl-diphosphate synthases also show similar patterns. These findings indicate that the elongating product passages on a surface of the side chains of the mutated amino acids, the original bulky amino acids had blocked the elongation, and the path is conserved in prenyltransferases. Moreover, the fact that some double and triple mutated enzymes can also form small amounts of products longer than C50 indicates that the paths in these mutated enzymes can partially access the outer surface of the enzymes.
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conversion from archaeal geranylgeranyl diphosphate synthase to farnesyl diphosphate synthase two amino acids before the first aspartate rich motif solely determine eukaryotic farnesyl diphosphate synthase activity
Journal of Biological Chemistry, 1997Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Kazutake Hirooka, Tokuzo NishinoAbstract:Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are precursors for a variety of important natural products, such as sterols, carotenoids, and prenyl quinones. Although FPP synthase and GGPP synthase catalyze similar consecutive condensations of isopentenyl diphosphate with allylic Diphosphates and have several homologous regions in their amino acid sequences, nothing is known about how these enzymes form the specific products. To locate the region that causes the difference of final products between GGPP synthase and FPP synthase, we constructed six mutated archaeal GGPP synthases whose regions around the first aspartate-rich motif were replaced with the corresponding regions of FPP synthases from human, rat, Arabidopsis thaliana, Saccharomyces cerevisiae, Escherichia coli, Bacillus stearothermophilus, and from some other related mutated enzymes. From the analysis of these mutated enzymes, we revealed that the region around the first aspartate-rich motif is essential for the product specificity of all FPP synthases and that the mechanism of the chain termination in eukaryotic FPP synthases (type I) is different from those of prokaryotic FPP synthases (type II). In FPP synthases of type I, two amino acids situated at the fourth and the fifth positions before the motif solely determine their product chain length, while the product specificity of the type II enzymes is determined by one aromatic amino acid at the fifth position before the motif, two amino acids inserted in the motif, and other modifications. These data indicate that FPP synthases have evolved from the progenitor corresponding to the archaeal GGPP synthase in two ways.
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a role of the amino acid residue located on the fifth position before the first aspartate rich motif of farnesyl diphosphate synthase on determination of the final product
Journal of Biological Chemistry, 1996Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Chika Ishida, Takeshi Nakazawa, Keishi Narita, Yoshie Takeuchi, Tokuzo NishinoAbstract:Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl Diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.
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Conversion of Product Specificity of Archaebacterial Geranylgeranyl-diphosphate Synthase IDENTIFICATION OF ESSENTIAL AMINO ACID RESIDUES FOR CHAIN LENGTH DETERMINATION OF PRENYLTRANSFERASE REACTION
The Journal of biological chemistry, 1996Co-Authors: Shin-ichi Ohnuma, Chikara Ohto, Chika Ishida, Kazutake Hirooka, Tokuzo NishinoAbstract:Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate with allylic Diphosphates to produce prenyl Diphosphates whose chain lengths are absolutely determined by each enzyme. To investigate the mechanism of the consecutive reaction and the determination of the ultimate chain length, a random mutational approach was planned. A geranylgeranyl-diphosphate synthase gene from Sulfolobus acidocaldarius was randomly mutagenized by NaNO2 treatment to construct a library of mutated geranylgeranyl-diphosphate synthase genes on a yeast expression vector. The library was screened for suppression of a pet phenotype of yeast C296-LH3, which is deficient in hexaprenyl-diphosphate synthase. Five mutants that could grow on a YEPG plate, which contained only glycerol as an energy source instead of glucose, were selected from approximately 1,400 mutants. All selected mutated enzymes catalyzed the formation of polyprenyl Diphosphates with prenyl chains longer than geranylgeranyl diphosphate. Especially mutants 1, 3, and 5 showed the strongest elongation activity to produce large amounts of geranylfarnesyl diphosphate with a concomitant amount of hexaprenyl diphosphate. Sequence analysis revealed that each mutant contained a few amino acid substitutions and that the mutation of Phe-77, which is located on the fifth amino acid upstream from the first aspartate-rich consensus motif, is the most effective for elongating the ultimate product. Amino acid alignment of known prenyltransferases around this position and our previous observations on farnesyl-diphosphate synthase (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T.(1996) J. Biol. Chem. 271, 10087-10095) clearly indicate that the amino acid at the position of all prenyltransferases must regulate the chain elongation.
Yungchi Cheng - One of the best experts on this subject based on the ideXlab platform.
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novel role of 3 phosphoglycerate kinase a glycolytic enzyme in the activation of l nucleoside analogs a new class of anticancer and antiviral agents
Journal of Biological Chemistry, 2003Co-Authors: Preethi Krishnan, Ginger E Dutschman, Elizabeth A Gullen, Susan P Grill, Yungchi ChengAbstract:Abstract l-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of l-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of l-nucleoside analogs (but not d-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of l-nucleotides in the cells, thereby playing a key role in the activity of l-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of l- and d-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog Diphosphates.
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phosphorylation of pyrimidine deoxynucleoside analog Diphosphates selective phosphorylation of l nucleoside analog Diphosphates by 3 phosphoglycerate kinase
Journal of Biological Chemistry, 2002Co-Authors: Preethi Krishnan, Qin Fu, Jiehyuan Liou, Ginger E Dutschman, Yungchi ChengAbstract:Abstract d-Nucleoside analogs, which are in the natural configuration, as well as the l-nucleoside analogs, are clinically relevant antiviral and anticancer agents. Metabolism of l-nucleoside analog Diphosphates to the triphosphates, however, remains unexplored. Studies with recombinant nm23-H1 and -H2 isoforms indicated that l-nucleoside analog Diphosphates were not phosphorylated by their nucleoside diphosphate kinase (NDPK) activity. Therefore, roles of creatine kinase, 3-phosphoglycerate kinase, and pyruvate kinase were evaluated using preparations from commercial sources and human HepG2 cells. Phosphorylation of l-OddC, l-SddC,l-Fd4C, l-FMAU, and l-ddC were compared with d-deoxynucleoside analogs, AraC, dFdC, andd-FMAU, and d-dideoxynucleoside analogs, ddC and d4T. Results based on preparations from HepG2 cells showed thatl-nucleoside analog Diphosphates were selectively phosphorylated by 3-phosphoglycerate kinase, whereas,d-deoxynucleoside analog Diphosphates were phosphorylated by NDPK. Interestingly, ddCDP and d4TDP were substrates for creatine kinase, but were not phosphorylated by NDPK. In conclusion, it is proposed that specificity of the phosphorylating enzymes toward the nucleoside analog Diphosphates is dependent on the configuration of the analog (l or d) and the presence or absence of 3′-hydroxyl group in the sugar moiety. The enzymatic process of phosphorylation of l- and d-nucleoside analog Diphosphates is different in cells.