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Hashimoto T.,University of Tokyo | Hashimoto J.,Japan Biological Informatics Consortium | Teruya K.,Okinawa Institute of Advanced science | Hirano T.,Okinawa Institute of Advanced science | And 5 more authors.
Journal of the American Chemical Society | Year: 2015

Versipelostatin (VST) is an unusual 17-membered macrocyclic polyketide product that contains a spirotetronate skeleton. In this study, the entire VST biosynthetic gene cluster (vst) spanning 108 kb from Streptomyces versipellis 4083-SVS6 was identified by heterologous expression using a bacterial artificial chromosome vector. Here, we demonstrate that an enzyme, VstJ, catalyzes the stereoselective [4+2]-cycloaddition between the conjugated diene and the exocyclic olefin of a newly identified tetronate-containing intermediate to form the spirotetronate skeleton during VST biosynthesis. © 2014 American Chemical Society.

Komatsu M.,Kitasato University | Komatsu K.,Kitasato University | Koiwai H.,Kitasato University | Yamada Y.,Kitasato University | And 8 more authors.
ACS Synthetic Biology | Year: 2013

An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host. © 2013 American Chemical Society.

Tokunaga Y.,Japan Biological Informatics Consortium | Tokunaga Y.,University of Tokyo | Takeuchi K.,Japan National Institute of Advanced Industrial Science and Technology | Takahashi H.,Japan National Institute of Advanced Industrial Science and Technology | And 3 more authors.
Nature Structural and Molecular Biology | Year: 2014

Mitogen-activated protein kinases (MAPKs) are essential to intracellular signal transduction. MAPKs anchor their pathway-specific substrates through so-called 'docking interactions' at locations distal from the active site. Docking interactions ensure efficient substrate recognition, but their contribution to the kinase reaction itself remains unclear. Herein, we use solution NMR to analyze the interaction between dually phosphorylated, active human p38 and the C-terminal fragments of its substrate MK2. p38 phosphorylation and ATP loading collaboratively induce the active conformation; subsequently, p38 accommodates MK2 phosphoacceptor residues in its active site. The docking interaction enhances binding of ATP and the phosphoacceptor to p38, accelerating the phosphotransfer reaction. Thus, the docking interaction enhances p38 's enzymatic activity toward pathway-specific substrates allosterically as well as by the anchor effect. These findings clarify how MAPK cascades are organized in cells, even under ATP-depleted conditions often associated with environmental stress.

Takeuchi K.,Japan National Institute of Advanced Industrial Science and Technology | Tokunaga Y.,Japan Science and Technology Agency | Imai M.,Japan Biological Informatics Consortium | Takahashi H.,Yokohama City University | And 2 more authors.
Scientific Reports | Year: 2014

LmrR is a multidrug transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. However, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region remains largely unknown. Here, we structurally and dynamically characterized LmrR in the apo, compound-bound and promoter-bound states. The compound-binding site of LmrR exhibits ps- 1/4s dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition. Meanwhile, the compound binding induces redistribution of ps-ns dynamics to the allosteric sites, which entropically favors the high-Affinity recognition. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound-And promoter-bound states. Collectively, the data show how LmrR can dynamically exert its functions through promiscuous multi-target interactions, in a manner that cannot be understood by a static structural view.

Kyoto University, Japan National Institute of Advanced Industrial Science, Technology and Japan Biological Informatics Consortium | Date: 2013-06-26

Reprogramming substances capable of substituting for Klf4, selected from the group consisting of members of the IRX family (e.g., IRX6), members of the GLIS family (e.g., GLIS1), members of the PTX family (e.g., PITX2), DMRTB1, and nucleic acids that encode the same, are provided. Also provided are a method of producing iPS cells, comprising the step of introducing into a somatic cell both one or more kinds of the above-described nuclear reprogramming substances and a substance capable of inducing iPS cells from a somatic cell when combined with Klf4. Still also provided are iPS cells comprising an extraneous nucleic acid that encodes any one of the above-described nuclear reprogramming substances, that can be obtained by the method, and a method of producing somatic cells by inducing the iPS cells to differentiate.

Kyoto University, Japan Biological Informatics Consortium, Japan National Institute of Advanced Industrial Science and Technology | Date: 2011-02-16

Provided are a method of improving the efficiency of establishment of iPS cells, comprising the step of contacting one or more substances selected from the group consisting of members of the GLIS family (e.g., GLIS1) and nucleic acids that encode the same and one or more substances selected from the group consisting of members of the Klf family and nucleic acids that encode the same, with a somatic cell, an iPS cell comprising an exogenous nucleic acid that encodes a member of the GLIS family or a member of the Klf family, that can be obtained by the method, and a method of producing a somatic cell by inducing the differentiation of the iPS cell.

University of Tokyo and Japan Biological Informatics Consortium | Date: 2010-04-14

Provided is an apparatus by which a nucleic acid may be analyzed quickly and precisely. Specifically, provided is an apparatus for analysis of a nucleic acid including: a processing unit for creating, based on a total base composition of a nucleic acid, a plurality of base composition sets, creating a hierarchical structure in which the base composition sets are hierarchized in ascending order of a number of bases in a partial base composition at a terminal position, and further creating connection relations between each of the base composition sets, and another base composition set having, at the terminal position, a partial base composition obtained by adding one base to a partial base composition at the terminal position of each of the base composition sets; a processing unit for calculating, for each of the partial base compositions, a predicted mass value of a corresponding product ion, and imparting a weight to each of the base composition sets based on a comparison between the predicted mass value and an actual mass value of a product ion derived from the nucleic acid; and a processing unit for selecting one of base composition sets belonging to each hierarchy while following the connection relations from an outermost hierarchy in the hierarchical structure, based on a weight imparted to each of the base composition sets, to thereby determine a base sequence sequentially from a terminal base of the nucleic acid.

Maruyama C.,Fukui Prefectural University | Toyoda J.,Fukui Prefectural University | Kato Y.,Toyama Prefectural University | Izumikawa M.,Japan Biological Informatics Consortium | And 5 more authors.
Nature Chemical Biology | Year: 2012

The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery. © 2012 Nature America, Inc. All rights reserved.

Japan National Institute of Advanced Industrial Science, Technology and Japan Biological Informatics Consortium | Date: 2013-09-18

Induced pluripotent stem cells having genetic information identical to that of a patient and yet having nature close to those of ES cells are prepared from human peripheral blood monocytes without leaving those genes in the resultant cells after use for their preparation. Reprogramming genes are loaded on sustained expression-inducing Sendai viral vectors which do not have an activity for integrating foreign genetic information into chromosomes and they are introduced into peripheral blood-derived monocytes to be expressed. Subsequently, the vector genome RNA comprising the reprogramming genes is removed from the cells to establish induced pluripotent stem cells. The above-described Sendai viral vector is capable of extremely simple and efficient preparation of induced pluripotent stem cells having genetic information identical to that of an individual who supplied the differentiated cell and yet this vector is safe presenting only a low risk of tumorigenesis. By supplying useful induced pluripotent stem cells, this Sendai viral vector can serve as a powerful tool in various applications including safe and efficient performance of cell replacement therapy.

Uchikoga N.,Japan Biological Informatics Consortium | Uchikoga N.,Tokyo Science and Industrial Research Institute | Hirokawa T.,Tokyo Science and Industrial Research Institute
BMC Bioinformatics | Year: 2010

Background: Protein-protein docking for proteins with large conformational changes was analyzed by using interaction fingerprints, one of the scales for measuring similarities among complex structures, utilized especially for searching near-native protein-ligand or protein-protein complex structures. Here, we have proposed a combined method for analyzing protein-protein docking by taking large conformational changes into consideration. This combined method consists of ensemble soft docking with multiple protein structures, refinement of complexes, and cluster analysis using interaction fingerprints and energy profiles.Results: To test for the applicability of this combined method, various CaM-ligand complexes were reconstructed from the NMR structures of unbound CaM. For the purpose of reconstruction, we used three known CaM-ligands, namely, the CaM-binding peptides of cyclic nucleotide gateway (CNG), CaM kinase kinase (CaMKK) and the plasma membrane Ca2+ATPase pump (PMCA), and thirty-one structurally diverse CaM conformations. For each ligand, 62000 CaM-ligand complexes were generated in the docking step and the relationship between their energy profiles and structural similarities to the native complex were analyzed using interaction fingerprint and RMSD. Near-native clusters were obtained in the case of CNG and CaMKK.Conclusions: The interaction fingerprint method discriminated near-native structures better than the RMSD method in cluster analysis. We showed that a combined method that includes the interaction fingerprint is very useful for protein-protein docking analysis of certain cases. © 2010 Uchikoga and Hirokawa; licensee BioMed Central Ltd.

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