KINDAI UNIVERSITY


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ISHIKAWA Fumihiro

Profile

FacultyDepartment of Pharmacy
PositionLecturer
DegreePh.D.in Science
Commentator Guidehttps://www.kindai.ac.jp/meikan/2073-ishikawa-fumihiro.html
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Last Updated :2020/08/10

Education and Career

Education

  •   2000 04  - 2004 03 , Meijo University, Faculty of Pharmacy
  •   2004 04  - 2006 03 , Osaka Prefecture University
  •   2006 04  - 2009 03 , Osaka Prefecture University, Graduate School of Science

Academic & Professional Experience

  •   2017 04 ,  - 現在, Faculty of Pharmacy, Kindai University
  •   2010 04 ,  - 2011 11 , University of California, San Diego
  •   2009 04 ,  - 2010 03 , Graduate School of Science, Osaka Prefecture University

Research Activities

Published Papers

  • Discovery of new benzhydrol biscarbonate esters as potent and selective apoptosis inducers of human melanomas bearing the activated ERK pathway: SAR studies on an ERK MAPK signaling modulator, ACA-28, Ryosuke Satoh, Naoya Hamada, Ami Yamada, Yuki Kanda, Fumihiro Ishikawa, Teruaki Takasaki, Genzoh Tanabe,* Reiko Sugiura*, Bioorg. Chem., Bioorg. Chem., Jul. 2020 , Refereed
  • Probing the compatibility of an enzyme-linked immunosorbent assay toward the reprogramming of nonribosomal peptide synthetase adenylation domains, Fumihiro Ishikawa,* Maya Nohara, Katsuki Takashima, Genzoh Tanabe*, ChemBioChem DOI: 10.1002/cbic.2000206, ChemBioChem DOI: 10.1002/cbic.2000206, Jun. 2020 , Refereed
  • Precise Probing of Residue Roles by NRPS Code Swapping: Mutation, Enzymatic Characterization, Modeling, and Substrate Promiscuity of Aryl Acid Adenylation Domains, Fumihiro Ishikawa,* Maya Nohara, Shinya Nakamura, Isao Nakanishi, Genzoh Tanabe*, Jan. 2020 , Refereed
  • Chemical strategies for visualizing and analyzing endogenous nonribosomal peptide synthetase (NRPS) megasynthetases, Fumihiro Ishikawa,* Genzoh Tanabe*, May 2019 , Refereed
  • Facile Synthesis of Neokotalanol, a Potent α‑glycosidase Inhibitor Isolated from the Ayurvedic Traditional Medicine “Salacia”, Genzoh Tanabe,* Satoshi Ueda, Kazuho Kurimoto, Naoki Sonoda, Shinsuke Marumoto, Fumihiro Ishikawa, Weijia Xie, Osamu Muraoka, 4, 7533 - 7542, Apr. 2019 , Refereed
  • An engineered aryl acid adenylation domain with an enlarged substrate binding pocket, Fumihiro Ishikawa,* Akimasa Miyanaga, Hinano Kitayama, Shinya Nakamura, Isao Nakanishi, Fumitaka Kudo, Tadashi Eguchi, Genzoh Tanabe*, Apr. 2019 , Refereed
  • Synthesis of salacinol-d4 as an internal standard for mass-spectrometric quantitation of salacinol, a potent α-glucosidase inhibitor found in a traditional ayurvedic medicine “Salacia”, Genzoh Tanabe,* Sanami Teramae, Yousuke Kunikata, Shuhei Okugawa, Shinsuke Marumoto, Fumihiro Ishikawa, Weija Xie, Toshio Morikawa, Osamu Muraoka, Sep. 2018 , Refereed
  • Expanding the Scope of Functionalized Small Nonprotein Components for Holoabzyme 27C1, Fumihiro Ishikawa, Masato Shirahashi, Hiroshi Hayakawa, Genzoh Tanabe, Takeshi Tsumuraya, Ikuo Fujii*, 3, 9313 - 9317, Aug. 2018 , Refereed
  • Structural basis of protein–protein interactions between a trans-acting acyltransferase and acyl carrier protein in polyketide disorazole biosynthesis, Akimasa Miyanaga,* Ouchi Risako, Fumihiro Ishikawa, Ena Goto, Genzoh Tanabe, Fumitaka Kudo, Tadashi Eguchi*, 140, 7970 - 7978, Jun. 2018 , Refereed
  • Activity-based protein profiling of non-ribosomal peptide synthetases, Fumihiro Ishikawa,* Genzoh Tanabe, Hideaki Kakeya*, 2018 , Refereed
  • Total Synthesis of γ-Alkylidenebutenolides, Potent Melanogenesis Inhibitors from Thai Medicinal Plant Melodorum fruticosum, Genzoh Tanabe,* Yoshiaki Manse, Teppei Ogawa, Naoki Sonoda, Shinsuke Marumoto, Fumihiro Ishikawa, Kiyofumi Ninomiya, Saowanee Chaipech, Yutana Pongpiriyadacha, Osamu Muraoka, Toshio Morikawa*, Journal of Organic Chemistry, Journal of Organic Chemistry, 83(15), 8250 - 8264, 2018 , Refereed
  • Diastereoselective synthesis of salacinol-type α-glucosidase inhibitors, Fumihiro Ishikawa, Kazumi Jinno, Naoki Sonoda, Eri Kinouchi, Kiyofumi Ninomiya, Shinsuke Marumoto, Weijia Xie, Osamu Muraoka, Toshio Morikawa, Genzoh Tanabe*, 83, 185 - 193, Dec. 2017 , Refereed
  • Visualizing the Adenylation Activities and Protein-Protein Interactions of Aryl Acid Adenylating Enzymes, Fumihiro Ishikawa* Shota Kasai, Hideaki Kakeya, Genzoh Tanabe*, CHEMBIOCHEM, CHEMBIOCHEM, 18(22), 2199 - 2204, Nov. 2017 , Refereed
    Summary:Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid Adomains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.
  • A Chemoproteomics Approach to Investigate Phosphopantetheine Transferase Activity at the Cellular Level, Sho Konno, Fumihiro Ishikawa,* Takehiro Suzuki, Naoshi Dohmae, Hideaki Kakeya, Genzoh Tanabe*, CHEMBIOCHEM, CHEMBIOCHEM, 18(18), 1855 - 1862, Sep. 2017 , Refereed
    Summary:Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.
  • Total syntheses of the aromatase inhibitors, mammeasins C and D, from Thai medicinal plant Mammea siamensis, Genzoh Tanabe,* Nozomi Tsutsui, Kanae Shibatani, Shinsuke Marumoto, Fumihiro Ishikawa, Kiyofumi Ninomiya, Osamu Muraoka, Toshio Morikawa*, TETRAHEDRON, TETRAHEDRON, 73(30), 4481 - 4486, Jul. 2017 , Refereed
    Summary:The first total syntheses of the geranylated pyranocoumarins, mameasins C (1) and D (2), aromatase inhibitors isolated from the flowers of Mammea siamensis, were accomplished in five steps, starting from phloroglucinol 3. In this strategy, the characteristic pyran ring-fused coumarin core of 1 and 2 was effectively constructed by Friedel-Crafts acylation of 3, followed by Reformatsky reaction of the resultant ketone to give a key coumarin intermediate 9. Compound 9 was converted to targets 1 and 2 in a stepwise manner by successive C-acylation and O-geranylation, followed by a [1,3]-sigmatropic geranyl shift. Furthermore, screening of intermediates obtained in the synthetic pathway to 1 and 2 revealed that de-geranylated pyranocoumarins (10 and 11) show superior aromatase inhibitory activity as compared to the natural products 1 and 2. (C) 2017 Elsevier Ltd. All rights reserved.
  • The chemical biology of natural product biosynthesis: chemical tools for the proteomic analysis of nonribosomal peptide synthetases, Fumihiro Ishikawa,* Hideaki Kakeya*, Frontiers in Nat. Prod. Chem., Frontiers in Nat. Prod. Chem., 3, 67 - 93, 2017 , Refereed
  • In Vitro Investigation of Crosstalk between Fatty Acid and Polyketide Synthases in the Andrimid Biosynthetic Assembly Line, Fumihiro Ishikawa,* Hiroyasu Sugimoto, Hideaki Kakeya*, CHEMBIOCHEM, CHEMBIOCHEM, 17(22), 2137 - 2142, Nov. 2016 , Refereed
    Summary:Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.
  • Site-Directed Chemical Mutations on Abzymes: Large Rate Accelerations in the Catalysis by Exchanging the Functionalized Small Nonprotein Components, Fumihiro Ishikawa, Masato Shirahashi, Hiroshi Hayakawa, Asako Yamaguchi, Takatsugu Hirokawa, Takeshi Tsumuraya, Ikuo Fujii*, ACS CHEMICAL BIOLOGY, ACS CHEMICAL BIOLOGY, 11(10), 2803 - 2811, Oct. 2016 , Refereed
    Summary:Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as site-directed chemical mutation. In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, beta-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of beta-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the beta-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg(H52) in 27C1 makes a hydrogen bond with the P-O oxygen in the hapten. This suggested that during beta-elimination, Arg(H52) of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the beta-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [(k(cat)/Km(2))/kuncat] of 1?000?000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg(H52) is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic pKa of the amine is perturbed and shifts to a lower pKa, which efficiently abstracts the alpha-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research.
  • A Competitive Enzyme-Linked Immunosorbent Assay System for Adenylation Domains in Nonribosomal Peptide Synthetases, Fumihiro Ishikawa,* Hideaki Kakeya*, CHEMBIOCHEM, CHEMBIOCHEM, 17(6), 474 - 478, Mar. 2016 , Refereed
    Summary:We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.
  • A chemical proteomic probe for detecting native carrier protein motifs in nonribosomal peptide synthetases, Shota Kasai, Fumihiro Ishikawa,* Takehiro Suzuki, Naoshi Dohmae, Hideaki Kakeya*, CHEMICAL COMMUNICATIONS, CHEMICAL COMMUNICATIONS, 52(98), 14129 - 14132, 2016 , Refereed
    Summary:Derivatization of a 5 '-(vinylsulfonylaminodeoxy) adenosine scaffold with a clickable functionality provided an activity-based probe that was used to label native carrier protein (CP) motifs in nonribosomal peptide synthetases (NRPSs). When coupled with a fluorescent tag, this probe selectively targeted phosphopantetheinylated CPs (holo-form) from recombinant NRPS enzyme systems and in whole proteomes.
  • Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains, Fumihiro Ishikawa,* Hideaki Kakeya*, Methods Mol. Biol., Methods Mol. Biol., 1401, 63 - 76, 2016 , Refereed
  • A Multiple-Labeling Strategy for Nonribosomal Peptide Synthetases Using Active-Site-Directed Proteomic Probes for Adenylation Domains, Fumihiro Ishikawa,* Takehiro Suzuki, Naoshi Dohmae, Hideaki Kakeya*, CHEMBIOCHEM, CHEMBIOCHEM, 16(18), 2590 - 2594, Dec. 2015 , Refereed
    Summary:Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments.
  • Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains, Fumihiro Ishikawa,* Kengo Miyamoto, Sho Konno, Shota Kasai, Hideaki Kakeya*, ACS CHEMICAL BIOLOGY, ACS CHEMICAL BIOLOGY, 10(12), 2816 - 2826, Dec. 2015 , Refereed
    Summary:A significant, gap exists between protein engineering and enzymes used for the biosynthesis of natural, products, largely because 1;, there is a paucity of strategies:that rapidly detect. active site phenotypes the enzymes with desired activities. Herein, we describe a proof-oft : concept study of an enzyme-linked immunosorbe4-asaay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetase (NRPSs)' using a,combination of active site-directed probes coupled a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality, that, immobilizes probe xriOlectles onto a Streptavidin-coated solid Support. The recombinant NRPSs have a C-terrinnal His tag motif that is targeted by an anti-6xHis mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected domains in the crude cell-free homogenates from the Escherichia,colt expression systems, When coupled with a chromogenic substrate, the antibody based ELISA technique, can :visualize probe protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenxoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal) activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the k(cat)/K-m value with the noncognate substrate Sal and a corresponding 48 fold decrease in the k(cat)/K-m Value with the cognate substrate DHB. The resulting 26 fold switch in substrate: specificity was achieved by the replacement. of,a Ser residue in the active site of EntE :with a Cys toward the nonribosomal codes of Sal-activating enzymes: Bringing a laboratory ELISA,technique and adenylating enzymes together using a combination of active site directed probes for the A domains in NRPSs should accelerate both the functional,characterization and manipulation of the A domains in NRPSs.
  • Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains, Fumihiro Ishikawa,* Sho Konno, Takehiro Suzuki, Naoshi Dohmae, Hideaki Kakeya*, ACS CHEMICAL BIOLOGY, ACS CHEMICAL BIOLOGY, 10(9), 1989 - 1997, Sep. 2015 , Refereed
    Summary:Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5'-O-N-(aminoacyl)sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production.
  • Recent Advances in Adenylation Domain Enzymology in Nonribosomal Peptide Biosynthesis, Fumihiro Ishikawa,* Hideaki Kakeya*, CURRENT ORGANIC CHEMISTRY, CURRENT ORGANIC CHEMISTRY, 19(13), 1204 - 1221, 2015 , Refereed
    Summary:Microorganisms produce a large number of peptide-based natural products that display a broad range of biologically interesting properties, including antimicrobial, immunosuppressant, and anticancer activities, as well as behaving as virulence factors and signaling molecules. These peptide natural products are composed of proteinogenic amino acids, as well as a number of other compound classes, including non-proteinogenic amino acids, aryl acids, fatty acids, hydroxyl acids, heterocyclic rings, and sugars, which provide a complex level of chemical diversity. Many of these natural products are biosynthesized by large, highly versatile multifunctional megasynthetases, which are known as nonribosomal peptide synthetases (NRPSs). The adenylation (A) domains found in all NRPS modules are essential catalytic components and function as gatekeepers to select the appropriate amino acid building blocks during nonribosomal peptide (NRP) biosynthesis. The results of extensive periods of genetic, biochemical, and bioinformatic investigations have provided a detailed understanding of the functional characteristics and molecular basis underpinning the A domain enzymology in NRP biosynthesis. This review will therefore focus on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the A domains within NRPS enzymes.
  • Functional profiling of adenylation domains in nonribosomal peptide synthetases by competitive activity-based protein profiling, Shota Kasai, Sho Konno, Fumihiro Ishikawa,* Hideaki Kakeya*, CHEMICAL COMMUNICATIONS, CHEMICAL COMMUNICATIONS, 51(87), 15764 - 15767, 2015 , Refereed
    Summary:We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.
  • Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases, Sho Konno, Fumihiro Ishikawa,* Takehiro Suzuki, Naoshi Dohmae, Michael D. Burkart, Hideaki Kakeya*, CHEMICAL COMMUNICATIONS, CHEMICAL COMMUNICATIONS, 51(12), 2262 - 2265, 2015 , Refereed
    Summary:We describe a general strategy for selective chemical labeling of individual adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using active site-directed proteomic probes coupled to the 5 '-O-N-(aminoacyl) sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.
  • Biosynthetic Origins of the Epoxyquinone Skeleton in Epoxyquinols A and B, Katsuki Fujita, Fumihiro Ishikawa, Hideaki Kakeya*, JOURNAL OF NATURAL PRODUCTS, JOURNAL OF NATURAL PRODUCTS, 77(12), 2707 - 2710, Dec. 2014 , Refereed
  • A 7-dimethylallyl tryptophan synthase from a fungal Neosartorya sp.: Biochemical characterization and structural insight into the regioselective prenylation, Kengo Miyamoto, Fumihiro Ishikawa, Shinya Nakamura, Yutaka Hayashi, Isao Nakanishi, Hideaki Kakeya*, BIOORGANIC & MEDICINAL CHEMISTRY, BIOORGANIC & MEDICINAL CHEMISTRY, 22(8), 2517 - 2528, Apr. 2014 , Refereed
    Summary:A putative 7-dimethylallyl tryptophan synthase (DMATS) gene from a fungal Neosartorya sp. was cloned and overexpressed as a soluble His(6)-fusion protein in Escherichia coli. The enzyme was found to catalyze the prenylation of L-tryptophan at the C7 position of the indole moiety in the presence of dimethylallyl diphosphate; thus, it functions as a 7-DMATS. In this study, we describe the biochemical characterization of 7-DMATS from Neosartorya sp., referred to as 7-DMATS(Neo), and the structural basis of the regioselective prenylation of L-tryptophan at the C7 position by comparison of the three-dimensional structural models of 7-DMATS(Neo) with FgaPT2 (4-DMATS) from Aspergillus fumigatus. (C) 2014 Elsevier Ltd. All rights reserved.
  • Specific enrichment of nonribosomal peptide synthetase module by an affinity probe for adenylation domains, Fumihiro Ishikawa,* Hideaki Kakeya*, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, 24(3), 865 - 869, Feb. 2014 , Refereed
    Summary:We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5'-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated L-Phe-AMS (L-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates L-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes. (C) 2014 Elsevier Ltd. All rights reserved.
  • Trapping the dynamic acyl carrier protein in fatty acid biosynthesis, Chi Nguyen, Robert W. Haushalter, D. John Lee, Phineus R. L. Markwick, Joel Bruegger, Grace Caldara-Festin, Kara Finzel, David R. Jackson, Fumihiro Ishikawa, Bing O'Dowd, J. Andrew McCammon, Stanley J. Opella, Shiou-Chuan Tsai,* Michael D. Burkart*, NATURE, NATURE, 505(7483), 427 - 431, Jan. 2014 , Refereed
    Summary:Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis(1). Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain(2). ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway(3-5). The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 angstrom crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.
  • Sulfonyl 3-Alkynyi Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase, Fumihiro Ishikawa, Robert W. Haushalter, D. John Lee, Kara Finzel, Michael D. Burkart*, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 135(24), 8846 - 8849, Jun. 2013 , Refereed
    Summary:Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein-protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic beta-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein-protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the crypto-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges.
  • Antibody-catalyzed decarboxylation and aldol reactions using a primary amine molecule as a functionalized small nonprotein component, Fumihiro Ishikawa, Kouki Uno, Masao Nishikawa, Takeshi Tsumuraya, Ikuo Fujii*, BIOORGANIC & MEDICINAL CHEMISTRY, BIOORGANIC & MEDICINAL CHEMISTRY, 21(22), 7011 - 7017, 2013 , Refereed
  • Dehydratase-Specific Probes for Fatty Acid and Polyketide Synthases, Fumihiro Ishikawa, Robert W. Haushalter, Michael D. Burkart*, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 134(2), 769 - 772, Jan. 2012 , Refereed
    Summary:We targeted the development of a dehydratase (DH)-specific reactive probe that can facilitate detection, enrichment, and identification of DH enzymes in fatty acid synthases (FASs) and polyketide synthases (PKSs). The first reported mechanism-based inactivator, 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), while active against the Escherichia coli beta-hydroxydecanoyl thiol ester DH FabA, translates poorly to an activity-based probe because of nonspecific reactivity of the thioester moiety. Here we describe the design, synthesis, and utility of a DH-specific probe that contains a sulfonyl 3-alkyne reactive warhead engineered to avoid hydrolysis or nonenzymatic inactivation. When coupled with a fluorescent tag, this probe targets DH enzymes from recombinant type I and type II FAS and PKS enzyme systems and in whole proteomes. Activity studies, including FabA inactivation and antibiotic susceptibility, suggest that this sulfonyl 3-alkyne scaffold selectively targets a common DH mechanism. These studies indicate that the DH-specific mechanism-based probe can greatly accelerate both the functional characterization and molecular identification of virtually any type of FAS and PKS in complex proteomes.
  • A Single Antibody Catalyzes Multiple Chemical Transformations upon Replacement of the Functionalized Small Nonprotein Components, Fumihiro Ishikawa, Takeshi Tsumuraya, Ikuo Fujii*, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 131(2), 456 - 467, Jan. 2009 , Refereed
    Summary:A single antibody catalyzes multiple chemical transformations upon replacement of the funcctionalized small nonportein components.

Conference Activities & Talks

  • Facile synthesis of neokotalanol, a potent α-glycosidase inhibitor isolated from, Genzoh Tanabe, Satoshi Ueda, Kazuho Kurimoto, Naoki Sonoda, Shinsuke Marumoto, Fumihiro Ishikawa, Osamu Muraoka,   2019 09
  • Reprogramming aryl acid adenylation domains for non-native building blocks, Fumihiro Ishikawa, Akimasa Miyanaga, Hinano Kitayama, Fumitaka Kudo, Tadashi Eguchi, Genzoh Tanabe,   2019 08

Misc

  • タイ天然薬物Mammea siamensis花部クマリン成分のCYP19阻害活性, 二宮清文, 二宮清文, LUO Fenglin, 柴谷華苗, CHAIPECH Saowanee, CHAIPECH Saowanee, PONGPIRIYADACHA Yutana, 村岡修, 石川文洋, 田邉元三, 田邉元三, 森川敏生, 森川敏生, 天然薬物の開発と応用シンポジウム講演要旨集, 22nd, 129‐131,   2018 10 01 , http://jglobal.jst.go.jp/public/201802213440413492
  • 4,5‐ジデヒドロアポルフィン型アルカロイドの合成およびメラニン形成抑制活性評価, 白戸美希, 萬瀬貴昭, 二宮清文, 丸本真輔, 石川文洋, 村岡修, 森川敏生, 田邉元三, 田邉元三, 日本薬学会年会要旨集(CD-ROM), 138th, 2, ROMBUNNO.27PA‐pm093, 186,   2018 03 , http://jglobal.jst.go.jp/public/201802257656280994
  • チオ糖とエポキシドとのS‐アルキル化を鍵反応に用いる“サラシア”由来,サラシノール型α‐グルコシダーゼ阻害剤の高ジアステレオ選択的合成, 石川文洋, 神農佳澄, 薗田直樹, 村岡修, 田邉元三, 田邉元三, 反応と合成の進歩シンポジウム講演要旨集, 43rd, 74,   2017 10 16 , http://jglobal.jst.go.jp/public/201702213296716806
  • “キャビコール誘導体ACA‐28”は,がん細胞特異的にERK依存的細胞死を誘導する革新的抗がん剤シーズである, 杉浦麗子, 佐藤亮介, 松浦一貴, 萩原加奈子, 神田勇輝, 石川文洋, 田邉元三, 村岡修, 高崎輝恒, メディシナルケミストリーシンポジウム講演要旨集, 35th, 80,   2017 10 04 , http://jglobal.jst.go.jp/public/201802227707522943
  • 天然薬物”サラシア”由来サラシノール類縁体のジアステレオ選択的合成及びin vivo α‐グルコシダーゼ阻害活性評価, 石川文洋, 神農佳澄, 薗田直樹, 木内恵里, 赤木淳二, 二宮清文, 村岡修, 吉川雅之, 森川敏生, 田邉元三, 田邉元三, メディシナルケミストリーシンポジウム講演要旨集, 35th, 252,   2017 10 04 , http://jglobal.jst.go.jp/public/201802280565741383
  • タイ天然薬物Melodorum fruticosum含有butenolide類のメラニン産生抑制活性, 萬瀬貴昭, 田邉元三, 二宮清文, 二宮清文, 今川貴仁, 安藤恵里, 福田梨沙, 福田友紀, 石川文洋, 村岡修, 村岡修, 森川敏生, 森川敏生, 日本生薬学会年会講演要旨集, 64回, 122, 122,   2017 08 , http://jglobal.jst.go.jp/public/201802254505833102