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Masahiro Okada - One of the best experts on this subject based on the ideXlab platform.
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A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto.
Chembiochem : a European journal of chemical biology, 2018Co-Authors: Tomotoshi Sugita, Masahiro Okada, Yu Nakashima, Tian Tian, Ikuro AbeAbstract:Bacillus subtilis subsp. natto secretes the ComXnatto pheromone as a quorum-sensing pheromone to produce poly-γ-glutamate for biofilm formation. The amino-acid sequence of the pheromone is Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue is post-translationally modified with a Farnesyl Group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C-terminal end of the precursor peptide ComXnatto . Here, we report the functional analysis of ComQnatto , which catalyzes a unique Farnesyl-transfer reaction. ComQnatto recognizes not only full-length ComXnatto but also N- and/or C-terminal truncated ComXnatto analogues and even a single tryptophan for modification with a Farnesyl Group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQnatto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.
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Posttranslational isoprenylation of tryptophan in bacteria.
Beilstein Journal of Organic Chemistry, 2017Co-Authors: Masahiro Okada, Tomotoshi Sugita, Ikuro AbeAbstract:Posttranslational isoprenylation is generally recognized as a universal modification of the cysteine residues in peptides and the thiol Groups of proteins in eukaryotes. In contrast, the Bacillus quorum sensing peptide pheromone, the ComX pheromone, possesses a posttranslationally modified tryptophan residue, and the tryptophan residue is isoprenylated with either a geranyl or Farnesyl Group at the gamma position to form a tricyclic skeleton that bears a newly formed pyrrolidine, similar to proline. The post-translational dimethylallylation of two tryptophan residues of a cyclic peptide, kawaguchipeptin A, from cyanobacteria has also been reported. Interestingly, the modified tryptophan residues of kawaguchipeptin A have the same scaffold as that of the ComX pheromones, but with the opposite stereochemistry. This review highlights the biosynthetic pathways and posttranslational isoprenylation of tryptophan. In particular, recent studies on peptide modifying enzymes are discussed.
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Identification of a quorum sensing pheromone posttranslationally Farnesylated at the internal tryptophan residue from Bacillus subtilis subsp. natto
Bioscience biotechnology and biochemistry, 2015Co-Authors: Shunsuke Hayashi, Syohei Usami, Yuta Nakamura, Koki Ozaki, Masahiro OkadaAbstract:Bacillus subtilis subsp. natto produces poly-γ-glutamic acid under the control of quorum sensing. We identified ComXnatto pheromone as the quorum-sensing pheromone with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu and the tryptophan residue posttranslationally modified by a Farnesyl Group. ComXnatto pheromone is unique in the sense that the 5th tryptophan residue from the C-terminal is Farnesylated.
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Chemical structure and biological activity of a quorum sensing pheromone from Bacillus subtilis subsp. natto
Bioorganic & medicinal chemistry letters, 2015Co-Authors: Masahiro Okada, Shunsuke Hayashi, Yuta Nakamura, Koki Ozaki, Syohei UsamiAbstract:Bacillus subtilis subsp. natto secrets a peptide pheromone, named ComXnatto pheromone, as an inducer for biofilm formation containing poly-γ-glutamic acid. Recently, the ComXnatto pheromone was identified to be a hexapeptide with an amino acid sequence of Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue was post-translationally modified with a Farnesyl Group. In order to determine the precise modification of the tryptophan residue, ComXnatto pheromone was synthesized using solid-phase peptide synthesis. Biological activity of the ComXnatto pheromone was then investigated. It was demonstrated that poly-γ-glutamic acid production were accelerated by ComXnatto pheromone at more than 1 nM in natto.
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Posttranslational Isoprenylation of Tryptophan Residues in Bacillus subtilis
Protein Prenylation PART A, 2011Co-Authors: Masahiro Okada, Fumitada Tsuji, Youji SakagamiAbstract:Publisher Summary Bacillus subtilis and related bacilli produce a posttranslationally modified oligopeptide termed “ComX pheromone” that stimulates natural genetic competence controlled by quorum sensing. ComX pheromones are modified with either a geranyl or Farnesyl Group on a conserved tryptophan residue at the 3-position of its indole ring, resulting in the formation of a tricyclic structure that includes a newly formed five-membered ring similar to proline. ComX pheromone is the first example of not only isoprenoidal modification of tryptophan residues in living organisms but also posttranslational isoprenylation in prokaryotes. As the geranylation of peptides—including monoterpene metabolites—is unusual in prokaryotes, posttranslational geranylation is unprecedented in nature. The modification of ComX pheromones with isoprenoid plays a more essential role for establishing genetic competence than the amino acid sequence. Based on the initial discovery of posttranslational isoprenylation of cysteine in pheromones secreted by eukaryotic microorganisms, which was later revealed to be a widespread phenomenon, it is speculated that posttranslational isoprenylation of tryptophan will also be recognized as a universal modification and will respond to important biological function in the near future.
Wenning Wang - One of the best experts on this subject based on the ideXlab platform.
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Lipid Regulated Conformational Dynamics of the Longin SNARE Protein Ykt6 Revealed by Molecular Dynamics Simulations
2015Co-Authors: Jingwei Weng, Yanhong Yang, Wenning WangAbstract:The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the Farnesylated state of Ykt6. In this study, we performed in silico Farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the Farnesylated Ykt6 model structure is stable during the 2 μs simulation and the Farnesyl Group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and Farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study
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Lipid Regulated Conformational Dynamics of the Longin SNARE Protein Ykt6 Revealed by Molecular Dynamics Simulations
The journal of physical chemistry. A, 2014Co-Authors: Jingwei Weng, Yanhong Yang, Wenning WangAbstract:The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the Farnesylated state of Ykt6. In this study, we performed in silico Farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the Farnesylated Ykt6 model structure is stable during the 2 μs simulation and the Farnesyl Group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and Farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study.
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Lipid-Induced Conformational Switch Controls Fusion Activity of Longin Domain SNARE Ykt6
Molecular cell, 2010Co-Authors: Wenyu Wen, Lifeng Pan, Zhiyi Wei, Jingwei Weng, Wenning Wang, Yan Shan Ong, Ton Hoai Thi Tran, Wanjin Hong, Mingjie ZhangAbstract:While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon Farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached Farnesyl Group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.
Ikuro Abe - One of the best experts on this subject based on the ideXlab platform.
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Multidomain P450 Epoxidase and a Terpene Cyclase from the Ascochlorin Biosynthetic Pathway in Fusarium sp.
Organic letters, 2019Co-Authors: Zhiyang Quan, Takayoshi Awakawa, Dongmei Wang, Ikuro AbeAbstract:Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the Farnesyl Group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated. Precursor-directed biosynthesis generated novel bromo-substituted derivatives, which exhibited potent cytotoxic activities. This study paves the way for the future metabolic engineering of medicinally important meroterpenoids for drug discovery.
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Multidomain P450 Epoxidase and a Terpene Cyclase from the Ascochlorin Biosynthetic Pathway in Fusarium sp.
2019Co-Authors: Zhiyang Quan, Takayoshi Awakawa, Dongmei Wang, Ikuro AbeAbstract:Ascochlorin is a medicinally important fungal meroterpenoid. Its biosynthetic pathway in Fusarium sp. was identified, and the stereoselective epoxidation of the Farnesyl Group by the multidomain, soluble P450 monooxygenase AscE and the subsequent formation of the unique timethylcyclohexanone ring by the membrane-bound cyclase AscF were investigated. Precursor-directed biosynthesis generated novel bromo-substituted derivatives, which exhibited potent cytotoxic activities. This study paves the way for the future metabolic engineering of medicinally important meroterpenoids for drug discovery
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A Tryptophan Prenyltransferase with Broad Substrate Tolerance from Bacillus subtilis subsp. natto.
Chembiochem : a European journal of chemical biology, 2018Co-Authors: Tomotoshi Sugita, Masahiro Okada, Yu Nakashima, Tian Tian, Ikuro AbeAbstract:Bacillus subtilis subsp. natto secretes the ComXnatto pheromone as a quorum-sensing pheromone to produce poly-γ-glutamate for biofilm formation. The amino-acid sequence of the pheromone is Lys-Trp-Pro-Pro-Ile-Glu, and the tryptophan residue is post-translationally modified with a Farnesyl Group to form a tricyclic scaffold. Unlike other Bacillus ComX pheromones, the tryptophan residue is distant from the C-terminal end of the precursor peptide ComXnatto . Here, we report the functional analysis of ComQnatto , which catalyzes a unique Farnesyl-transfer reaction. ComQnatto recognizes not only full-length ComXnatto but also N- and/or C-terminal truncated ComXnatto analogues and even a single tryptophan for modification with a Farnesyl Group in vitro. These results, together with the calculated kinetic parameters, suggest that ComQnatto does not require a leader sequence for substrate recognition and is a promising enzyme with broad substrate tolerance for the synthesis of various prenylated tryptophan derivatives.
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Posttranslational isoprenylation of tryptophan in bacteria.
Beilstein Journal of Organic Chemistry, 2017Co-Authors: Masahiro Okada, Tomotoshi Sugita, Ikuro AbeAbstract:Posttranslational isoprenylation is generally recognized as a universal modification of the cysteine residues in peptides and the thiol Groups of proteins in eukaryotes. In contrast, the Bacillus quorum sensing peptide pheromone, the ComX pheromone, possesses a posttranslationally modified tryptophan residue, and the tryptophan residue is isoprenylated with either a geranyl or Farnesyl Group at the gamma position to form a tricyclic skeleton that bears a newly formed pyrrolidine, similar to proline. The post-translational dimethylallylation of two tryptophan residues of a cyclic peptide, kawaguchipeptin A, from cyanobacteria has also been reported. Interestingly, the modified tryptophan residues of kawaguchipeptin A have the same scaffold as that of the ComX pheromones, but with the opposite stereochemistry. This review highlights the biosynthetic pathways and posttranslational isoprenylation of tryptophan. In particular, recent studies on peptide modifying enzymes are discussed.
Fuyuhiko Tamanoi - One of the best experts on this subject based on the ideXlab platform.
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Farnesylated proteins and cell cycle progression.
Journal of Cellular Biochemistry, 2002Co-Authors: Fuyuhiko Tamanoi, Juran Kato-stankiewicz, Iara M. P. Machado, Chen Jiang, Nitika ThaparAbstract:Post-translational modification of proteins by the addition of a Farnesyl Group is critical for the function of a number of proteins involved in signal transduction. Farnesylation facilitates their membrane association and also promotes protein-protein interaction. Recently, progress has been made in understanding the biological significance of Farnesylation. First, effects of Farnesyltransferase inhibitors (FTIs) on cancer cells have been examined using a variety of human cancer cells. This study showed that one of the major effects of FTIs is to alter cell cycle progression. Both G0/G1 enrichment and G2/M accumulation were observed depending on the cell line examined. Second, a number of novel Farnesylated proteins have been characterized. Of these, Rheb and CENP-E,F are of particular interest. Rheb, a novel member of the Ras superfamily G-proteins, may play a role in the G1 phase of the cell cycle. CENP-E,F are centromere associated motors that play critical roles in mitosis. These results suggest important contributions of Farnesylated proteins in the regulation of cell cycle progression.
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Genetic Analysis of FTase and GGTase I and Natural Product Farnesyltransferase Inhibitors
2001Co-Authors: Fuyuhiko Tamanoi, Keith Del Villar, Nicole G. G. Robinson, Meerhan Kim, Jun Urano, Wenli YangAbstract:Protein prenyltransferases are conserved from yeast to humans (1,2). Because yeast and human enzymes are structurally and functionally similar, efforts have been made to use yeast as a genetic system to study protein prenyltransferases. Recently, the yeast system was used to obtain a number of Farnesyltransferase (FTase) mutants that provided valuable information concerning residues of FTase important for substrate recognition and catalysis (3,4). The yeast system’ s usefulness for the study of protein prenylation was initially established from the study of the yeast mating factor, a-factor (5,6). This short peptide is modified by the addition of a Farnesyl Group. The structure determination of this peptide provided insights into the chemical nature of the modification as well as other processing events that accompany Farnesylation. The study on the processing of yeast Ras proteins also provided critical information on Farnesylation and related processing events, and subsequently led to the identification of the yeast genes encoding FTase (1,7).
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Prenylation of RAS and Inhibitors of Prenyltransferases
Regulation of the RAS Signaling Network, 1996Co-Authors: Isabel Sattler, Fuyuhiko TamanoiAbstract:The recent development of Farnesyltransferase inhibitors which inhibit the growth of RAS-transformed cells demonstrates that membrane association of RAS and other monomeric G proteins such as Rho is important for the tumorigenicity of RAS.1–3 In the early 1980s, it was established that RAS is C-terminally modified and that this modification is important for both its membrane association and tumorigenicity. This observation was followed by the elucidation of the steps involved in the C-terminal modification. The first step in this modification is the addition of a Farnesyl Group to RAS utilizing Farnesyl pyrophosphate (FPP) as the prenyl donor. Characterization of the Farnesylation led to the identification of the enzyme Farnesyltransferase (FTase). This enzyme and two related enzymes, geranylgeranyltransferases (GGTases) I and II, form a family of enzymes called protein prenyl-transferases. The structure and function of these enzymes are being elucidated, which could provide vital information concerning the active sites of the enzyme. Inhibitors of these enzymes have been identified by a variety of approaches, and the inhibitors have been utilized to inhibit the growth of RAS tumors. In this chapter we will focus on two topics: (i) the structure and function of protein prenyltransferases and (ii) inhibitors of prenyltransferases.
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[5] In vivo assays for Farnesyltransferase inhibitors with Saccharomyces cereuisiae
Methods in enzymology, 1995Co-Authors: Hiroshi Mitsuzawa, Fuyuhiko TamanoiAbstract:Publisher Summary This chapter describes simple plate assays for Farnesyltransferase inhibitors using yeast gpal and RAS2Val-9 mutants. The methods are useful in screening natural products for inhibitors and in evaluating synthetic inhibitors. Protein Farnesylation is important for protein function. The modification is catalyzed by Farnesyltransferase, which transfers a Farnesyl Group from Farnesyl diphosphate to a cysteine residue located in a carboxyl-terminal tetrapeptide sequence (the CaaX motif) of an acceptor protein. Farnesylated proteins include Ras proteins, nuclear lamins, the γ subunits of transducin and yeast heterotrimeric G protein, and fungal mating pheromones. Farnesyltransferase inhibitors provide valuable tools for the understanding of the catalytic mechanism of the enzyme. The assays are used to evaluate synthetic inhibitors such as peptidomimetics because the yeast system provides simple in vivo assays. It is possible that the effects of inhibitors are different between the yeast and mammalian enzymes and that the penetration and degradation of inhibitors are different between yeast and mammalian cells.
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s Farnesylation and methyl esterification of c terminal domain of yeast ras2 protein prior to fatty acid acylation
Journal of Biological Chemistry, 1991Co-Authors: Asao Fujiyama, Susumu Tsunasawa, Fuyuhiko Tamanoi, Fumio SakiyamaAbstract:Abstract Posttranslational processing/modification is required for membrane localization and activation of ras proteins. In the case of yeast RAS2 protein, we have reported that the process starts with the removal of the initiator methionine followed by polyisoprenylation, removal of 3 amino acid residues from the C terminus, methyl esterification, and fatty acid acylation (Fujiyama, A., and Tamanoi, F. (1990) J. Biol. Chem. 265, 3362-3368). In this study, we demonstrate that polyisoprenylation and methyl esterification of the cysteine residue in the C-terminal domain of the RAS2 protein are involved in the conversion process from precursor form to intermediate form. The polyisoprenoid moiety attached to the RAS2 protein was identified as a 15-carbon Farnesyl Group through two independent experiments: the release of S-Farnesylcysteine with carboxypeptidase Y from the RAS2 protein, and the recovery of radioactive farnesol through methyliodide treatment of the RAS2 protein purified from yeast cells labeled with [3H]mevalonic acid. The Farnesyl Group attached to the RAS2 protein was detected predominantly in the C-terminal peptide, SGSGGCC, both in the intermediate and in the fatty acid acylated RAS2 protein. The C-terminal cysteine of the intermediate protein is also modified by methyl esterification in a nearly stoichiometric manner.
Jingwei Weng - One of the best experts on this subject based on the ideXlab platform.
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Lipid Regulated Conformational Dynamics of the Longin SNARE Protein Ykt6 Revealed by Molecular Dynamics Simulations
2015Co-Authors: Jingwei Weng, Yanhong Yang, Wenning WangAbstract:The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the Farnesylated state of Ykt6. In this study, we performed in silico Farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the Farnesylated Ykt6 model structure is stable during the 2 μs simulation and the Farnesyl Group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and Farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study
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Lipid Regulated Conformational Dynamics of the Longin SNARE Protein Ykt6 Revealed by Molecular Dynamics Simulations
The journal of physical chemistry. A, 2014Co-Authors: Jingwei Weng, Yanhong Yang, Wenning WangAbstract:The conformation and subcellular localization of R-SNARE protein Ykt6 are regulated by the lipidation state of its C-terminal CCAIM motif. Biochemical and crystallography studies showed that lipid molecules binding at a hydrophobic pocket at the interface between the longin domain and the SNARE core can lock Ykt6 at a closed conformation and mimic the Farnesylated state of Ykt6. In this study, we performed in silico Farnesylation of Ykt6 and explored the conformational dynamics of Ykt6 using conventional and steered MD simulations. We found that the Farnesylated Ykt6 model structure is stable during the 2 μs simulation and the Farnesyl Group adopts conformations similar to those of the DPC molecule bound to Ykt6. Both DPC binding and Farnesylation were found to reduce the conformational flexibility of Ykt6 and hinder the dissociation of SNARE core from the longin domain. The dissociation of the αF-αG segment is the rate-limiting step during the putative closed-to-open conformational transition of Ykt6, and the key residues involved in this process are consistent with the experimental mutagenesis study.
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Lipid-Induced Conformational Switch Controls Fusion Activity of Longin Domain SNARE Ykt6
Molecular cell, 2010Co-Authors: Wenyu Wen, Lifeng Pan, Zhiyi Wei, Jingwei Weng, Wenning Wang, Yan Shan Ong, Ton Hoai Thi Tran, Wanjin Hong, Mingjie ZhangAbstract:While most SNAREs are permanently anchored to membranes by their transmembrane domains, the dually lipidated SNARE Ykt6 is found both on intracellular membranes and in the cytosol. The cytosolic Ykt6 is inactive due to the autoinhibition of the SNARE core by its longin domain, although the molecular basis of this inhibition is unknown. Here, we demonstrate that unlipidated Ykt6 adopts multiple conformations, with a small population in the closed state. The structure of Ykt6 in complex with a fatty acid suggests that, upon Farnesylation, the Ykt6 SNARE core forms four alpha helices that wrap around the longin domain, forming a dominantly closed conformation. The fatty acid, buried in a hydrophobic groove formed between the longin domain and its SNARE core, is essential for maintaining the autoinhibited conformation of Ykt6. Our study reveals that the posttranslationally attached Farnesyl Group can actively regulate Ykt6 fusion activity in addition to its anticipated membrane-anchoring role.
