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Tokuzo Nishino - One of the best experts on this subject based on the ideXlab platform.
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The product chain length determination mechanism of type II geranylgeranyl Diphosphate synthase requires subunit interaction.
The FEBS journal, 2008Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi HemmiAbstract: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. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl Diphosphate synthase.
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Short-chain Prenyl Diphosphate synthase that condenses isopentenyl Diphosphate with dimethylallyl Diphosphate in ispA null Escherichia coli strain lacking farnesyl Diphosphate synthase.
Journal of bioscience and bioengineering, 2007Co-Authors: Ken Saito, Shingo Fujisaki, Tokuzo NishinoAbstract:A short-chain Prenyl Diphosphate synthase in an Escherichia coli mutant that lacked the gene coding for farnesyl Diphosphate synthase, ispA, was separated from other Prenyl Diphosphate synthases by DEAE-Toyopearl column chromatography. The purified enzyme catalyzed the condensation of isopentenyl Diphosphate with dimethylallyl Diphosphate to form farnesyl Diphosphate and geranylgeranyl Diphosphate.
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isolation and expression of paracoccus denitrificans decaPrenyl Diphosphate synthase gene for production of ubiquinone 10 in escherichia coli
Biochemical Engineering Journal, 2003Co-Authors: Seiji Takahashi, Tokuzo Nishino, Tanetoshi KoyamaAbstract:Abstract Ubiquinones (coenzyme Q) play important roles as an electron carrier in the mitochondrial respiratory chain and have been successfully used as an orally administrated prophylaxis and therapy for various diseases. The number of isoprene unit in the Prenyl side chain of ubiquinone varies depending on the organism. This organism-dependency of the isoprene unit number is determined by the availability of the Prenyl Diphosphate in the cell. Paracoccus denitrificans has (all-E)-decaPrenyl Diphosphate (DecPP) synthase catalyzing condensation of seven molecules of isopentenyl Diphosphate with (all-E)-farnesyl Diphosphate to afford DecPP (C50), the precursor of the Prenyl side chain of coenzyme Q-10. To understand structure–activity relationship of Prenyl chain elongating enzymes in molecular level as well as to establish a manipulation system of ubiquinone-10 in Escherichia coli cells, the structural gene encoding DecPP synthase was cloned from Paracoccus denitrificans. An overproducing system of this enzyme was constructed, and the Prenyltransferase assay of the cell-free system of the transformant indicated that the recombinant protein overexpressed in E. coli cells showed distinct DecPP synthase activity. Moreover, the level of ubiquinone-10 in the transformed cells was greater than that of ubiquinone-8, which is intrinsic in wild type E. coli host cells.
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an alternative mechanism of product chain length determination in type iii geranylgeranyl Diphosphate synthase
FEBS Journal, 2003Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, 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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An alternative mechanism of product chain‐length determination in type III geranylgeranyl Diphosphate synthase
European journal of biochemistry, 2003Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, 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.
Hisashi Hemmi - One of the best experts on this subject based on the ideXlab platform.
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Utilization of an intermediate of the methylerythritol phosphate pathway, (E)-4-hydroxy-3-methylbut-2-en-1-yl Diphosphate, as the Prenyl donor substrate for various Prenyltransferases.
Bioscience biotechnology and biochemistry, 2018Co-Authors: Yoshifumi Hayashi, Tohru Yoshimura, Tomokazu Ito, Hisashi HemmiAbstract:Abstract(E)-4-hydroxy-3-methylbut-2-en-1-yl Diphosphate (HMBPP) is an intermediate of the methylerythritol phosphate pathway. Utilization of HMBPP by lycopene elongase from Corynebacterium glutamicum, which is a UbiA-family Prenyltransferase responsible for C50 carotenoid biosynthesis, was investigated using an Escherichia coli strain that contained the exogenous mevalonate pathway as well as the carotenoid biosynthetic pathway. Inhibition of the endogenous methylerythritol phosphate pathway resulted in loss of the production of C50 carotenoid flavuxanthin, while C40 lycopene formation was retained. Overexpression of E. coli ispH gene, which encodes HMBPP reductase, also decreased the production of flavuxanthin in E. coli cells. These results indicate the preference of lycopene elongase for HMBPP instead of the previously proposed substrate, dimethylallyl Diphosphate. Furthermore, several (all-E)-Prenyl Diphosphate synthases, which are classified in a distinct family of Prenyltransferase, were demonstrate...
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A cis‐Prenyltransferase from Methanosarcina acetivorans catalyzes both head‐to‐tail and nonhead‐to‐tail Prenyl condensation
The FEBS journal, 2016Co-Authors: Takuya Ogawa, Tohru Yoshimura, Koh-ichi Emi, Kazushi Koga, Hisashi HemmiAbstract:Cis-Prenyltransferase usually consecutively catalyzes the head-to-tail condensation reactions of isopentenyl Diphosphate to allylic Prenyl Diphosphate in the production of (E,Z-mixed) polyPrenyl Diphosphate, which is the precursor of glycosyl carrier lipids. Some recently discovered homologs of the enzyme, however, catalyze the nonhead-to-tail condensation reactions between allylic Prenyl Diphosphates. In this study, we characterize a cis-Prenyltransferase homolog from a methanogenic archaeon, Methanosarcina acetivorans, to obtain information on the biosynthesis of the glycosyl carrier lipids within it. This enzyme catalyzes both head-to-tail and nonhead-to-tail condensation reactions. The kinetic analysis shows that the main reaction of the enzyme is consecutive head-to-tail Prenyl condensation reactions yielding polyPrenyl Diphosphates, while the chain lengths of the major products seem shorter than expected for the precursor of glycosyl carrier lipids. On the other hand, a subsidiary reaction of the enzyme, i.e., nonhead-to-tail condensation between dimethylallyl Diphosphate and farnesyl Diphosphate, gives a novel diterpenoid compound, geranyllavandulyl Diphosphate.
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Geranylfarnesyl Diphosphate synthase from Methanosarcina mazei: Different role, different evolution
Biochemical and Biophysical Research Communications, 2010Co-Authors: Takuya Ogawa, Tohru Yoshimura, Hisashi HemmiAbstract:The gene of (all-E) geranylfarnesyl Diphosphate synthase that is responsible for the biosynthesis of methanophenazine, an electron carrier utilized for methanogenesis, was cloned from a methanogenic archaeon Methanosarcina mazei Go1. The properties of the recombinant enzyme and the results of phylogenetic analysis suggest that the enzyme is closely related to (all-E) Prenyl Diphosphate synthases that are responsible for the biosynthesis of respiratory quinones, rather than to the enzymes involved in the biosynthesis of archaeal membrane lipids, including (all-E) geranylfarnesyl Diphosphate synthase from a thermophilic archaeon.
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The product chain length determination mechanism of type II geranylgeranyl Diphosphate synthase requires subunit interaction.
The FEBS journal, 2008Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi HemmiAbstract: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. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl Diphosphate synthase.
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an alternative mechanism of product chain length determination in type iii geranylgeranyl Diphosphate synthase
FEBS Journal, 2003Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, 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.
Tanetoshi Koyama - One of the best experts on this subject based on the ideXlab platform.
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Product chain-length determination mechanism of Z,E-farnesyl Diphosphate synthase.
Biochemical and biophysical research communications, 2008Co-Authors: Motoyoshi Noike, Sebabrata Mahapatra, Dean C. Crick, Takanori Ambo, Sayaka Kikuchi, Toshihide Suzuki, Satoshi Yamashita, Seiji Takahashi, Hirofumi Kurokawa, Tanetoshi KoyamaAbstract: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. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer Prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-Prenyltransferase, Micrococcus luteus B-P 26 undecaPrenyl Diphosphate synthase also resulted in the production of different Prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-Prenyltransferases.
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The product chain length determination mechanism of type II geranylgeranyl Diphosphate synthase requires subunit interaction.
The FEBS journal, 2008Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi HemmiAbstract: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. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl Diphosphate synthase.
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isolation and expression of paracoccus denitrificans decaPrenyl Diphosphate synthase gene for production of ubiquinone 10 in escherichia coli
Biochemical Engineering Journal, 2003Co-Authors: Seiji Takahashi, Tokuzo Nishino, Tanetoshi KoyamaAbstract:Abstract Ubiquinones (coenzyme Q) play important roles as an electron carrier in the mitochondrial respiratory chain and have been successfully used as an orally administrated prophylaxis and therapy for various diseases. The number of isoprene unit in the Prenyl side chain of ubiquinone varies depending on the organism. This organism-dependency of the isoprene unit number is determined by the availability of the Prenyl Diphosphate in the cell. Paracoccus denitrificans has (all-E)-decaPrenyl Diphosphate (DecPP) synthase catalyzing condensation of seven molecules of isopentenyl Diphosphate with (all-E)-farnesyl Diphosphate to afford DecPP (C50), the precursor of the Prenyl side chain of coenzyme Q-10. To understand structure–activity relationship of Prenyl chain elongating enzymes in molecular level as well as to establish a manipulation system of ubiquinone-10 in Escherichia coli cells, the structural gene encoding DecPP synthase was cloned from Paracoccus denitrificans. An overproducing system of this enzyme was constructed, and the Prenyltransferase assay of the cell-free system of the transformant indicated that the recombinant protein overexpressed in E. coli cells showed distinct DecPP synthase activity. Moreover, the level of ubiquinone-10 in the transformed cells was greater than that of ubiquinone-8, which is intrinsic in wild type E. coli host cells.
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Artificial substrates of medium-chain elongating enzymes, hexaPrenyl- and heptaPrenyl Diphosphate synthases.
Bioorganic & medicinal chemistry letters, 2001Co-Authors: Masahiko Nagaki, Tokuzo Nishino, Kosei Kimura, Hiroaki Kimura, Yuji Maki, Eiji Goto, Tanetoshi KoyamaAbstract:We examined the reactivity of 3-alkyl group homologues of farnesyl Diphosphate or isopentenyl Diphosphate for medium-chain Prenyl Diphosphate synthases, hexaPrenyl Diphosphate- or heptaPrenyl Diphosphate synthase. But-3-enyl Diphosphate, which lacks the methyl group at the 3-position of isopentenyl Diphosphate, condensed only once with farnesyl Diphosphate to give E-norgeranylgeranyl Diphosphate by the action of either enzyme. However, norfarnesyl Diphosphate was never accepted as an allylic substrate at all. 3-Ethylbut-3-enyl Diphosphate also reacted with farnesyl Diphosphate giving a mixture of (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl- and (all-E)-3,7-diethyl-11,15,19-trimethylicosa-2,6,10,14,18-pentaenyl Diphosphates by hexaPrenyl Diphosphate synthase. On the other hand, heptaPrenyl Diphosphate synthase reaction of 3-ethylbut-3-enyl Diphosphate with farnesyl Diphosphate gave only (all-E)-3-ethyl-7,11,15-trimethylhexadeca-2,6,10,14-tetraenyl Diphosphate.
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Chain length determination of Prenyltransferases: both heteromeric subunits of medium-chain (E)-Prenyl Diphosphate synthase are involved in the product chain length determination.
Biochemistry, 2000Co-Authors: Yuan-wei Zhang, Tanetoshi KoyamaAbstract:Among Prenyltransferases, medium-chain (E)-Prenyl Diphosphate synthases are unusual because of their heterodimeric structures. The larger subunit has highly conserved regions typical of (E)-Prenyltransferases. The smaller one has recently been shown to be involved in the binding of allylic substrate as well as determining the chain length of the reaction product [Zhang, Y.-W., et al. (1999) Biochemistry 38, 14638−14643]. To better understand the product chain length determination mechanism of these enzymes, several amino acid residues in the larger subunits of Micrococcus luteus B-P 26 hexaPrenyl Diphosphate synthase and Bacillus subtilis heptaPrenyl Diphosphate synthase were selected for substitutions by site-directed mutagenesis and examined by combination with the corresponding wild-type or mutated smaller subunits. Replacement of the Ala at the fifth position upstream to the first Asp-rich motif with bulky amino acids in both larger subunits resulted in shortening the chain lengths of the major produc...
David L. Gasser - One of the best experts on this subject based on the ideXlab platform.
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parkinson s disease like neuromuscular defects occur in Prenyl Diphosphate synthase subunit 2 pdss2 mutant mice
Mitochondrion, 2012Co-Authors: Carly G. K. Ziegler, Min Peng, Marni J. Falk, Erzsebet Polyak, Elpida Tsika, Harry Ischiropoulos, Dana Bakalar, Julie A. Blendy, David L. GasserAbstract:The Pdss2 gene product is needed for the isoPrenylation of benzoquinone to generate coenzyme Q (CoQ). A fatal kidney disease occurs in mice that are homozygous for a missense mutation in Pdss2, which can be recapitulated in conditional Pdss2 knockouts targeted to glomerular podocytes. We now report that homozygous missense mutants also demonstrate significant neuromuscular deficits, as validated by behavioral and coordination assays, and these deficits are recapitulated in conditional Pdss2 knockouts targeted to dopaminergic neurons. Both conditional knockout and missense mutant mice demonstrate deficiencies in tyrosine hydroxylase-positive neurons in the substantia nigra, implicating a pathology similar to sporadic Parkinson's disease (PD).
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Parkinson’s disease-like neuromuscular defects occur in Prenyl Diphosphate synthase subunit 2 (Pdss2) mutant mice
Mitochondrion, 2011Co-Authors: Carly G. K. Ziegler, Min Peng, Marni J. Falk, Erzsebet Polyak, Elpida Tsika, Harry Ischiropoulos, Dana Bakalar, Julie A. Blendy, David L. GasserAbstract:The Pdss2 gene product is needed for the isoPrenylation of benzoquinone to generate coenzyme Q (CoQ). A fatal kidney disease occurs in mice that are homozygous for a missense mutation in Pdss2, which can be recapitulated in conditional Pdss2 knockouts targeted to glomerular podocytes. We now report that homozygous missense mutants also demonstrate significant neuromuscular deficits, as validated by behavioral and coordination assays, and these deficits are recapitulated in conditional Pdss2 knockouts targeted to dopaminergic neurons. Both conditional knockout and missense mutant mice demonstrate deficiencies in tyrosine hydroxylase-positive neurons in the substantia nigra, implicating a pathology similar to sporadic Parkinson's disease (PD).
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Coenzyme Q10 supplementation rescues renal disease in Pdss2kd/kd mice with mutations in Prenyl Diphosphate synthase subunit 2
American journal of physiology. Renal physiology, 2008Co-Authors: Ryoichi Saiki, Adam L. Lunceford, Yuchen Shi, Beth N. Marbois, Rhonda King, Justin Pachuski, Makoto Kawamukai, David L. Gasser, Catherine F. ClarkeAbstract:Homozygous mice carrying kd (kidney disease) mutations in the gene encoding Prenyl Diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal...
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coenzyme q10 supplementation rescues renal disease in pdss2kd kd mice with mutations in Prenyl Diphosphate synthase subunit 2
American Journal of Physiology-renal Physiology, 2008Co-Authors: Ryoichi Saiki, Adam L. Lunceford, Yuchen Shi, Beth N. Marbois, Rhonda King, Justin Pachuski, Makoto Kawamukai, David L. Gasser, Catherine F. ClarkeAbstract:Homozygous mice carrying kd (kidney disease) mutations in the gene encoding Prenyl Diphosphate synthase subunit 2 (Pdss2kd/kd) develop interstitial nephritis and eventually die from end-stage renal...
Motoyoshi Noike - One of the best experts on this subject based on the ideXlab platform.
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Product chain-length determination mechanism of Z,E-farnesyl Diphosphate synthase.
Biochemical and biophysical research communications, 2008Co-Authors: Motoyoshi Noike, Sebabrata Mahapatra, Dean C. Crick, Takanori Ambo, Sayaka Kikuchi, Toshihide Suzuki, Satoshi Yamashita, Seiji Takahashi, Hirofumi Kurokawa, Tanetoshi KoyamaAbstract: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. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer Prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-Prenyltransferase, Micrococcus luteus B-P 26 undecaPrenyl Diphosphate synthase also resulted in the production of different Prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-Prenyltransferases.
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The product chain length determination mechanism of type II geranylgeranyl Diphosphate synthase requires subunit interaction.
The FEBS journal, 2008Co-Authors: Motoyoshi Noike, Tokuzo Nishino, Takashi Katagiri, Toru Nakayama, Tanetoshi Koyama, Hisashi HemmiAbstract: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. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechaism of chain length determination in type III geranylgeranyl Diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the α-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl Diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl Diphosphate synthase.
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an alternative mechanism of product chain length determination in type iii geranylgeranyl Diphosphate synthase
FEBS Journal, 2003Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, 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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An alternative mechanism of product chain‐length determination in type III geranylgeranyl Diphosphate synthase
European journal of biochemistry, 2003Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, 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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Change of product specificity of hexaPrenyl Diphosphate synthase from Sulfolobus solfataricus by introducing mimetic mutations
Biochemical and biophysical research communications, 2002Co-Authors: Hisashi Hemmi, Motoyoshi Noike, Toru Nakayama, Tokuzo NishinoAbstract:The introduction of several sets of amino acid substitutions into the region around a substrate-binding site of a medium-chain (all-E) Prenyl Diphosphate synthase, hexaPrenyl Diphosphate synthase from a thermoacidophilic archaeon Sulfolobus solfataricus, to mimic the product determination mechanisms of various kinds of short-chain enzymes revealed that the structure around the region of the medium-chain enzyme resembles those of eukaryotic farnesyl Diphosphate synthases but not those of the other short-chain enzymes, reflecting the evolutional relationships among these enzymes.