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Kazufumi Yazaki - One of the best experts on this subject based on the ideXlab platform.
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comparative proteomic analysis of lithospermum erythrorhizon reveals regulation of a variety of metabolic enzymes leading to comprehensive understanding of the shikonin biosynthetic pathway
Plant and Cell Physiology, 2019Co-Authors: Akifumi Sugiyama, Kojiro Takanashi, Yukimi Nakagawa, Shunsuke Aburaya, Kenta Kaminade, Wataru Aoki, Yuka Saidamunakata, Mitsuyoshi Ueda, Kazufumi YazakiAbstract:Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound Geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.
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homogeneous purification and characterization of lepgt1 a membrane bound aromatic substrate prenyltransferase involved in secondary metabolism of lithospermum erythrorhizon
FEBS Journal, 2013Co-Authors: Kazuaki Ohara, Koji Mito, Kazufumi YazakiAbstract:Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: Geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.
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Homogeneous purification and characterization of LePGT1 – a membrane‐bound aromatic substrate prenyltransferase involved in secondary metabolism of Lithospermum erythrorhizon
The FEBS journal, 2013Co-Authors: Kazuaki Ohara, Koji Mito, Kazufumi YazakiAbstract:Membrane-bound type prenyltransferases for aromatic substrates play crucial roles in the biosynthesis of various natural compounds. Lithospermum erythrorhizon p-hydroxybenzoate: Geranyltransferase (LePGT1), which contains multiple transmembrane α-helices, is involved in the biosynthesis of a red naphthoquinone pigment, shikonin. Taking LePGT1 as a model membrane-bound aromatic substrate prenyltransferase, we utilized a baculovirus-Sf9 expression system to generate a high yield LePGT1 polypeptide, reaching ~ 1000-fold higher expression level compared with a yeast expression system. Efficient solubilization procedures and biochemical purification methods were developed to extract LePGT1 from the membrane fraction of Sf9 cells. As a result, 80 μg of LePGT1 was purified from 150 mL culture to almost homogeneity as judged by SDS/PAGE. Using purified LePGT1, enzymatic characterization, e.g. substrate specificity, divalent cation requirement and kinetic analysis, was done. In addition, inhibition experiments revealed that aromatic compounds having two phenolic hydroxyl groups effectively inhibited LePGT1 enzyme activity, suggesting a novel recognition mechanism for aromatic substrates. As the first example of solubilization and purification of this membrane-bound protein family, the methods established in this study will provide valuable information for the precise biochemical characterization of aromatic prenyltransferases as well as for crystallographic analysis of this novel enzyme family.
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Characterization of coumarin-specific prenyltransferase activities in Citrus limon peel.
Bioscience biotechnology and biochemistry, 2012Co-Authors: Ryosuke Munakata, Tsuyoshi Inoue, Takao Koeduka, Kanako Sasaki, Yusuke Tsurumaru, Akifumi Sugiyama, Yoshihiro Uto, Hitoshi Hori, Jun-ichi Azuma, Kazufumi YazakiAbstract:Coumarins, a large group of polyphenols, play important roles in the defense mechanisms of plants, and they also exhibit various biological activities beneficial to human health, often enhanced by prenylation. Despite the high abundance of prenylated coumarins in citrus fruits, there has been no report on coumarin-specific prenyltransferase activity in citrus. In this study, we detected both O- and C-prenyltransferase activities of coumarin substrates in a microsome fraction prepared from lemon (Citrus limon) peel, where large amounts of prenylated coumarins accumulate. Bergaptol was the most preferred substrate out of various coumarin derivatives tested, and geranyl diphosphate (GPP) was accepted exclusively as prenyl donor substrate. Further enzymatic characterization of bergaptol 5-O-Geranyltransferase activity revealed its unique properties: apparent K m values for GPP (9 µM) and bergaptol (140 µM) and a broad divalent cation requirement. These findings provide information towards the discovery of a y...
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functional characterization of lepgt1 a membrane bound prenyltransferase involved in the geranylation of p hydroxybenzoic acid
Biochemical Journal, 2009Co-Authors: Kazuaki Ohara, Ayumu Muroya, Nobuhiro Fukushima, Kazufumi YazakiAbstract:The AS-PT (aromatic substrate prenyltransferase) family plays a critical role in the biosynthesis of important quinone compounds such as ubiquinone and plastoquinone, although biochemical characterizations of AS-PTs have rarely been carried out because most members are membrane-bound enzymes with multiple transmembrane α-helices. PPTs [PHB (p-hydroxybenzoic acid) prenyltransferases] are a large subfamily of AS-PTs involved in ubiquinone and naphthoquinone biosynthesis. LePGT1 [Lithospermum erythrorhizon PHB Geranyltransferase] is the regulatory enzyme for the biosynthesis of shikonin, a naphthoquinone pigment, and was utilized in the present study as a representative of membrane-type AS-PTs to clarify the function of this enzyme family at the molecular level. Site-directed mutagenesis of LePGT1 with a yeast expression system indicated three out of six conserved aspartate residues to be critical to the enzymatic activity. A detailed kinetic analysis of mutant enzymes revealed the amino acid residues responsible for substrate binding were also identified. Contrary to ubiquinone biosynthetic PPTs, such as UBIA in Escherichia coli which accepts many prenyl substrates of different chain lengths, LePGT1 can utilize only geranyl diphosphate as its prenyl substrate. Thus the substrate specificity was analysed using chimeric enzymes derived from LePGT1 and UBIA. In vitro and in vivo analyses of the chimeras suggested that the determinant region for this specificity was within 130 amino acids of the N-terminal. A 3D (three-dimensional) molecular model of the substrate-binding site consistent with these biochemical findings was generated.
Satish K. Nair - One of the best experts on this subject based on the ideXlab platform.
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
Journal of the American Chemical Society, 2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis.
Journal of the American Chemical Society, 2018Co-Authors: Maho Morita, Yue Hao, Jouni Jokela, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool provid...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis
2018Co-Authors: Maho Morita, Yue Hao, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Jouni K. Jokela, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C5 or C10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products
Lutz Heide - One of the best experts on this subject based on the ideXlab platform.
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A Membrane‐Bound Prenyltransferase Catalyzes the O‐Prenylation of 1,6‐Dihydroxyphenazine in the Marine Bacterium Streptomyces sp. CNQ‐509
Chembiochem : a European journal of chemical biology, 2014Co-Authors: Philipp Zeyhle, Judith S. Bauer, Marco Steimle, Franziska Leipoldt, Manuela Rösch, Jörn Kalinowski, Harald Gross, Lutz HeideAbstract:Streptomyces sp. CNQ-509 produces the rare O-prenylated phenazines marinophenazines A and B. To identify the enzyme catalyzing the O-prenyl transfer in marinophenazine biosynthesis, we sequenced the genome of S. sp. CNQ-509. This led to the identification of two genomic loci harboring putative phenazine biosynthesis genes. The first locus contains orthologues for all seven genes involved in phenazine-1-carboxylic acid biosynthesis in pseudomonads. The second locus contains two known phenazine biosynthesis genes and a putative prenyltransferase gene termed cnqPT1. cnqPT1 codes for a membrane protein with sequence similarity to the prenyltransferase UbiA of ubiquinone biosynthesis. The enzyme CnqPT1 was identified as a 1,6-dihydroxyphenazine Geranyltransferase, which catalyzes the CO bond formation between C-1 of the geranyl moiety and O-6 of the phenazine scaffold. CnqPT1 is the first example of a prenyltransferase catalyzing O-prenyl transfer to a phenazine.
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4-Hydroxybenzoate 3-Geranyltransferase from Lithospermum erythrorhizon: Purification of a plant membrane-bound prenyltransferase
Planta, 1998Co-Authors: Agnes Muhlenweg, Martin Melzer, Shu-ming Li, Lutz HeideAbstract:Geranyldiphosphate:4-hydroxybenzoate 3-Geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K(m) values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 microM and 13.8 microM, respectively.
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4-hydroxybenzoate prenyltransferases in cell-free extracts of Lithospermum erythrorhizon cell cultures
Phytochemistry, 1997Co-Authors: Robert Boehm, Martin Melzer, Lutz HeideAbstract:Abstract Aromatic prenylation reactions of 4-hydroxybenzoate (4HB) are involved in the biosynthesis of ubiquinones and of shikonin, a naphthoquinone pigment derived from 4HB and geranyldiphosphate (GPP) in Lithospermum erythrorhizon. The enzymic prenylation of 4HB with GPP and with solanesyldiphosphate (SPP) was measured in cell-free extracts from L. erythrorhizon cell cultures. The conversion of GPP was induced by methyl jasmonate, an inducer of shikonin biosynthesis, whilst the conversion of SPP was not, suggesting that the two reactions are carried out by different enzymes. Either reaction was found both in the microsomal fraction and in the organellar membrane fraction. The activity of the 4HB Geranyltransferase in the microsomal fraction could be separated from the 4HB solanesyltransferase by partial purification using DEAE Sephacel and Heparin Sepharose affinity columns. The results indicate the presence of two distinct enzymes, GPP:4-HB Geranyltransferase and SPP:4-HB solanesyltransferase. The enzymes are apparently quite specific for the chain length of the isoprenoid precursors, which is in contrast to the broad substrate specificity of the polyprenyldiphosphate: 4-HB polyprenyltransferase from Escherichia coli.
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inhibition and regulation of shikonin biosynthesis in suspension cultures of lithospermum
Phytochemistry, 1996Co-Authors: Sibylle Gaisser, Lutz HeideAbstract:Abstract The accumulation of acetylshikonin, p -hydroxybenzoic acid- O -glucoside (PHB-Glc) and rosmarinic acid (RA) was investigated in cell suspension cultures of Lithospermum erythrorhizon under the influence of blue and white light, of in vivo inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and phenylalanine ammonia lyase (PAL), and of the effectors methyl jasmonate and arsenate. The activities of p -hydroxybenzoate Geranyltransferase, p -hydroxybenzoate O -glucosyltransferase and HMG-CoA reductase were assayed. 2-Aminoindan-2-phosphonic acid, an inhibitor of PAL, completely suppressed the formation of acetylshikonin, PHB-Glc and RA. Mevinolin, an inhibitor of HMG-CoA reductase specifically blocked the formation of acetylshikonin, thereby increasing the formation of PHB-Glc. The activity of p -hydroxybenzoate- O -glucosyltransferase was not regulated by the intracellular concentration of p -hydroxybenzoate, but directly by light. White light rather than blue light induced the activity of this glucosyltransferase, whereas PHB Geranyltransferase activity was repressed by blue light. The activity of HMG-CoA reductase correlated with the production of acetylshikonin. This suggests a regulatory role for HMG-CoA reductase in the isoprenoid part of shikonin biosynthesis.
Eric W. Schmidt - One of the best experts on this subject based on the ideXlab platform.
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
Journal of the American Chemical Society, 2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis.
Journal of the American Chemical Society, 2018Co-Authors: Maho Morita, Yue Hao, Jouni Jokela, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool provid...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis
2018Co-Authors: Maho Morita, Yue Hao, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Jouni K. Jokela, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C5 or C10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products
Maho Morita - One of the best experts on this subject based on the ideXlab platform.
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
Journal of the American Chemical Society, 2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis.
Journal of the American Chemical Society, 2018Co-Authors: Maho Morita, Yue Hao, Jouni Jokela, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool provid...
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Post-Translational Tyrosine Geranylation in Cyanobactin Biosynthesis
2018Co-Authors: Maho Morita, Yue Hao, Debosmita Sardar, Zhenjian Lin, Kaarina Sivonen, Satish K. Nair, Jouni K. Jokela, Eric W. SchmidtAbstract:Prenylation is a widespread modification that improves the biological activities of secondary metabolites. This reaction also represents a key modification step in biosyntheses of cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by cyanobacteria. In cyanobactins, amino acids are commonly isoprenylated by ABBA prenyltransferases that use C5 donors. Notably, mass spectral analysis of piricyclamides from a fresh-water cyanobacterium suggested that they may instead have a C10 geranyl group. Here we characterize a novel Geranyltransferase involved in piricyclamide biosynthesis. Using the purified enzyme, we show that the enzyme PirF catalyzes Tyr O-geranylation, which is an unprecedented post-translational modification. In addition, the combination of enzymology and analytical chemistry revealed the structure of the final natural product, piricyclamide 7005E1, and the regioselectivity of PirF, which has potential as a synthetic biological tool providing drug-like properties to diverse small molecules
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A Single Amino Acid Switch Alters the Isoprene Donor Specificity in Ribosomally Synthesized and Post-Translationally Modified Peptide Prenyltransferases
2018Co-Authors: Paola Estrada, Maho Morita, Yue Hao, Eric W. Schmidt, Satish K. NairAbstract:Mutation at a single amino acid alters the isoprene donor specificity of prenyltransferases involved in the modification of ribosomally synthesized and post-translationally modified peptides (RiPPs). Though most characterized RiPP prenyltransferases carry out the regiospecific transfer of C5 dimethylallyl donor to the side chain atoms on macrocyclic acceptor substrates, the elucidation of the cyanobactin natural product piricyclamide 70005E1 identifies an O-geranyl modification on Tyr, a reaction with little prior biochemical precedence. Reconstitution and kinetic studies of the presumptive Geranyltransferase PirF shows that the enzyme utilizes a C10 donor, with no C5 transferase activity. The crystal structure of PirF reveals a single amino acid difference in the vicinity of the isoprene-binding pocket, relative to the C5 utilizing enzymes. Remarkably, only a single amino acid mutation is necessary to completely switch the donor specificity from a C5 to a C10 prenyltransferase, and vice versa. Lastly, we demonstrate that these enzymes may be used for the chemospecific attachment of C5 or C10 lipid groups on lanthipeptides, an unrelated class of RiPP natural products. These studies represent a rare example where prenyl donor specificity can be discretely altered, which expands the arsenal of synthetic biology tools for tuning biological activities of peptide natural products