Updated on 2024/02/02

写真a

 
KITAOKU Yoshihito
 
Organization
Research Institute for Interdisciplinary Science Special-Appointment Assistant Professor
Position
Special-Appointment Assistant Professor
External link

Degree

  • Doctor of Agricultural Science ( 2018.3   Kinki University )

Research Interests

  • GHs, Chitin, Chitosan, Chitinase, LysM, CBMs, bacterial two component system, Vibrios, membrane-trafficking, Asgard archaea, cytoskeleton, signal transduction, NMR, X-ray crystallography, biochemistry, structural biology, biophysics

Research Areas

  • Life Science / Structural biochemistry

Education

  • Kinki University   農学研究科   バイオサイエンス専攻

    2015.4 - 2018.3

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  • Kinki University   農学研究科   バイオサイエンス専攻

    2013.4 - 2015.3

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  • Kinki University   農学部  

    2009.4 - 2013.3

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    Country: Japan

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Research History

  • Okayama University   The Research Institute for Interdisciplinary Science

    2020.3

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    Country:Japan

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  • Vidyasirimedhi University of Science and Technology

    2019.3 - 2020.2

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    Country:Thailand

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  • Suranaree University of Technology

    2018.5 - 2019.1

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    Country:Thailand

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  • Japan Society for the Promotion of Science

    2016.4 - 2018.3

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Professional Memberships

  • 日本農芸化学会

    2022

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  • The Japanese Society of Applied Glycoscience

    2014

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  • 日本キチン・キトサン学会

    2014

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Papers

  • AlphaFold2: A versatile tool to predict the appearance of functional adaptations in evolution: Profilin interactions in uncultured Asgard archaea: Profilin interactions in uncultured Asgard archaea. International journal

    Khongpon Ponlachantra, Wipa Suginta, Robert C Robinson, Yoshihito Kitaoku

    BioEssays : news and reviews in molecular, cellular and developmental biology   e2200119   2022.12

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    The release of AlphaFold2 (AF2), a deep-learning-aided, open-source protein structure prediction program, from DeepMind, opened a new era of molecular biology. The astonishing improvement in the accuracy of the structure predictions provides the opportunity to characterize protein systems from uncultured Asgard archaea, key organisms in evolutionary biology. Despite the accumulation in metagenomics-derived Asgard archaea eukaryotic-like protein sequences, limited structural and biochemical information have restricted the insight in their potential functions. In this review, we focus on profilin, an actin-dynamics regulating protein, which in eukaryotes, modulates actin polymerization through (1) direct actin interaction, (2) polyproline binding, and (3) phospholipid binding. We assess AF2-predicted profilin structures in their potential abilities to participate in these activities. We demonstrate that AF2 is a powerful new tool for understanding the emergence of biological functional traits in evolution.

    DOI: 10.1002/bies.202200119

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  • Structure, mechanism, and phylogeny of LysM-chitinase conjugates specifically found in fern plants. International journal

    Yoshihito Kitaoku, Toki Taira, Tomoyuki Numata, Takayuki Ohnuma, Tamo Fukamizo

    Plant science : an international journal of experimental plant biology   321   111310 - 111310   2022.8

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    A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al., Biosci. Biotechnol. Biochem., 79, 1296-1304, 2015), using an Escherichia coli expression system and characterized for its structure and mechanism of action. The crystal structure of EaLysM exhibited an almost identical fold (βααβ) to that of PrLysM2. From isothermal titration calorimetry and nuclear magnetic resonance, the binding mode and affinities of EaLysM for chitooligosaccharides (GlcNAc)n (3, 4, 5, and 6) were found to be comparable to those of PrLysM2. The LysM module in EaChiA is likely to bind (GlcNAc)n almost independently through CH-π stacking of a Tyr residue with the pyranose ring. The (GlcNAc)n-binding mode of LysMs in the LysM-chitinase conjugates from fern plants appears to differ from that of plant LysMs acting in chitin- or Nod-signal perception, in which multiple LysMs cooperatively act on (GlcNAc)n. Phylogenetic analysis suggested that LysM-GH18 conjugates of fern plants formed a monophyletic group and had been separated earlier than forming the clade of fungal chitinases with LysMs.

    DOI: 10.1016/j.plantsci.2022.111310

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  • A structural model for (GlcNAc)2 translocation via a periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria. International journal

    Yoshihito Kitaoku, Tamo Fukamizo, Sawitree Kumsaoad, Prakayfun Ubonbal, Robert C Robinson, Wipa Suginta

    The Journal of biological chemistry   297 ( 3 )   101071 - 101071   2021.9

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    VhCBP is a periplasmic chitooligosaccharide-binding protein mainly responsible for translocation of the chitooligosaccharide (GlcNAc)2 across the double membranes of marine bacteria. However, structural and thermodynamic understanding of the sugar-binding/-release processes of VhCBP is relatively less. VhCBP displayed the greatest affinity toward (GlcNAc)2, with lower affinity for longer-chain chitooligosaccharides [(GlcNAc)3-4]. (GlcNAc)4 partially occupied the closed sugar-binding groove, with two reducing-end GlcNAc units extending beyond the sugar-binding groove and barely characterized by weak electron density. Mutation of three conserved residues (Trp363, Asp365, and Trp513) to Ala resulted in drastic decreases in the binding affinity toward the preferred substrate (GlcNAc)2, indicating their significant contributions to sugar binding. The structure of the W513A-(GlcNAc)2 complex in a 'half-open' conformation unveiled the intermediary step of the (GlcNAc)2 translocation from the soluble CBP in the periplasm to the inner membrane-transporting components. Isothermal calorimetry data suggested that VhCBP adopts the high-affinity conformation to bind (GlcNAc)2, while its low-affinity conformation facilitated sugar release. Thus, chitooligosaccharide translocation, conferred by periplasmic VhCBP, is a crucial step in the chitin catabolic pathway, allowing Vibrio bacteria to thrive in oceans where chitin is their major source of nutrients.

    DOI: 10.1016/j.jbc.2021.101071

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  • Mythical origins of the actin cytoskeleton. International journal

    Caner Akıl, Yoshihito Kitaoku, Linh T Tran, David Liebl, Han Choe, Duangkamon Muengsaen, Wipa Suginta, Albert Schulte, Robert C Robinson

    Current opinion in cell biology   68   55 - 63   2021.2

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    The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition.

    DOI: 10.1016/j.ceb.2020.08.011

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  • Molecular insight into a new low-affinity xylan binding module from the xylanolytic gut symbiont Roseburia intestinalis. International journal

    Maria Louise Leth, Morten Ejby, Eva Madland, Yoshihito Kitaoku, Dirk J Slotboom, Albert Guskov, Finn Lillelund Aachmann, Maher Abou Hachem

    The FEBS journal   287 ( 10 )   2105 - 2117   2020.5

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    Efficient capture of glycans, the prime metabolic resources in the human gut, confers a key competitive advantage for gut microbiota members equipped with extracellular glycoside hydrolases (GHs) to target these substrates. The association of glycans to the bacterial cell surface is typically mediated by carbohydrate binding modules (CBMs). Here, we report the structure of RiCBM86 appended to a GH family 10 xylanase from Roseburia intestinalis. This CBM represents a new family of xylan binding CBMs present in xylanases from abundant and prevalent healthy human gut Clostridiales. RiCBM86 adopts a canonical β-sandwich fold, but shows structural divergence from known CBMs. The structure of RiCBM86 has been determined with a bound xylohexaose, which revealed an open and shallow binding site. RiCBM86 recognizes only a single xylosyl ring with direct hydrogen bonds. This mode of recognition is unprecedented amongst previously reported xylan binding type-B CBMs that display more extensive hydrogen-bonding patterns to their ligands or employ Ca2+ to mediate ligand-binding. The architecture of RiCBM86 is consistent with an atypically low binding affinity (KD  about 0.5 mm for xylohexaose) compared to most xylan binding CBMs. Analyses using NMR spectroscopy corroborated the observations from the complex structure and the preference of RiCBM86 to arabinoxylan over glucuronoxylan, consistent with the largely negatively charged surface flanking the binding site. Mutational analysis and affinity electrophoresis established the importance of key binding residues, which are conserved in the family. This study provides novel insight into the structural features that shape low-affinity CBMs that mediate extended bacterial glycan capture in the human gut niche. DATABASES: Structural data are available in the protein data bank database under the accession number 6SGF. Sequence data are available in the GenBank database under the accession number EEV01588.1. The assignment of the Roseburia intestinalis xylan binding module into the CBM86 new family is available in the CAZy database (http://www.cazy.org/CBM86.html).

    DOI: 10.1111/febs.15117

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  • Novel GH-20 β-N-acetylglucosaminidase inhibitors: Virtual screening, molecular docking, binding affinity, and anti-tumor activity. International journal

    Piyanat Meekrathok, Sunisa Thongsom, Anuwat Aunkham, Anuphon Kaewmaneewat, Yoshihito Kitaoku, Kiattawee Choowongkomon, Wipa Suginta

    International journal of biological macromolecules   142   503 - 512   2020.1

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    β-N-acetylglucosaminidases (GlcNAcases) play a crucial role in the metabolism of glycan-conjugated proteins/lipids in humans. Elevated levels of serum GlcNAcases have been associated with certain types of cancer, and GlcNAcases therefore serve as drug targets. Here, we employed virtual screening to identify two novel GlcNAcase inhibitors from the National Cancer Institute (NCI) Drug Library using a bacterial GH-20 GlcNAcase (VhGlcNAcase) as a search model. NSC73735 was shown to be most potent with IC50 of 12.7 ± 1.2 μM, agreeing with Kd of 0.94 ± 0.2 μM obtained by ITC. Molecular docking refinement indicated that Trp582 the key residue that interacted with all the inhibitor molecules. Docking NSC7373 into the active site of human O-GlcNAcase (hOGA) yielded reasonably good fit with the estimated Kd of 44.7 µM, indicating its possibility to be a true binding partner. NSC73735 was shown to significantly suppress both cell growth and GlcNAcase activity of five cancer cell lines (U937, THP-1, MCF-7, HepG2 and PC-3) that express endogenous GlcNAcases. The cell cytotoxicity assay indicated the inherent effects of the lead compound on GlcNAcase expression with cancer cell proliferation, and therefore this novel GlcNAcase inhibitor may serve as a virtuous candidate for further development of highly potent anti-tumor agents.

    DOI: 10.1016/j.ijbiomac.2019.09.122

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  • Structures and chitin-binding properties of two N-terminal lysin motifs (LysMs) found in a chitinase from Volvox carteri. International journal

    Yoshihito Kitaoku, Shigenori Nishimura, Takeru Hirono, Wipa Suginta, Takayuki Ohnuma, Tamo Fukamizo

    Glycobiology   29 ( 7 )   565 - 575   2019.7

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    Two N-terminal lysin motifs (LysMs) found in a chitinase from the green alga Volvox carteri (VcLysM1 and VcLysM2) were produced, and their structures and chitin-binding properties were characterized. The binding affinities of VcLysM1 toward chitin oligomers determined by isothermal titration calorimetry (ITC) were higher than those of VcLysM2 by 0.8-1.1 kcal/mol of ΔG°. Based on the NMR solution structures of the two LysMs, the differences in binding affinities were found to result from amino acid substitutions at the binding site. The NMR spectrum of a two-domain protein (VcLysM1+2), in which VcLysM1 and VcLysM2 are linked in tandem through a flexible linker, suggested that the individual domains of VcLysM1+2 independently fold and do not interact with each other. ITC analysis of chitin-oligomer binding revealed two different binding sites in VcLysM1+2, showing no cooperativity. The binding affinities of the VcLysM1 domain in VcLysM1+2 were lower than those of VcLysM1 alone, probably due to the flexible linker destabilizing the interaction between the chito-oligosaccahrides and VcLysM1 domain. Overall, two LysMs attached to the chitinase from the primitive plant species, V. carteri, were found to resemble bacterial LysMs reported thus far.

    DOI: 10.1093/glycob/cwz024

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  • Periplasmic solute-binding proteins: Structure classification and chitooligosaccharide recognition. International journal

    Tamo Fukamizo, Yoshihito Kitaoku, Wipa Suginta

    International journal of biological macromolecules   128   985 - 993   2019.5

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    Periplasmic solute-binding proteins (SBPs) serve as molecular shuttles that assist the transport of small solutes from the outer membrane to the inner membrane of all Gram-negative bacteria. Based on the available crystal structures, SBPs are classified into seven clusters, A-G, and are further divided into subclusters, IV. This minireview is focused on the classification, structure and substrate specificity of a distinct class of SBPs specific for chitooligosaccharides (CBPs). To date, only two structures of CBP homologues, VhCBP and VcCBP, have been reported in the marine Vibrio species, with exposition of their limited function. The Vibrio CBPs are structurally classified as cluster C/subcluster IV SBPs that exclusively recognize β-1,4- or β-1,3-linked linear oligosaccharides. The overall structural feature of the Vibrios CBPs is most similar to the cellobiose-binding orthologue from the hyperthermophilic bacterium Thermotoga maritima. This similarity provides an opportunity to engineer the substrate specificity of the proteins and to control the uptake of chitinous and cellulosic nutrients in marine bacteria.

    DOI: 10.1016/j.ijbiomac.2019.02.064

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  • 1H, 13C and 15N backbone and side-chain assignment of a carbohydrate binding module from a xylanase from Roseburia intestinalis. International journal

    Eva Madland, Yoshihito Kitaoku, Gerd Inger Sætrom, Maria Louise Leth, Morten Ejby, Maher Abou Hachem, Finn Lillelund Aachmann

    Biomolecular NMR assignments   13 ( 1 )   55 - 58   2019.4

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    The N-terminal domain (residues 28-165) from the glycoside hydrolase family 10 from Roseburia intestinalis (RiCBMx), has been isotopically labeled and recombinantly expressed in Escherichia coli. Here we report 1H, 13C and 15N NMR chemical shift assignments for this carbohydrate binding module (CBM).

    DOI: 10.1007/s12104-018-9850-3

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  • Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A. International journal

    Yoshihito Kitaoku, Gaston Courtade, Dejan M Petrović, Tamo Fukamizo, Vincent G H Eijsink, Finn L Aachmann

    Biomolecular NMR assignments   12 ( 2 )   357 - 361   2018.10

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    The apo-form of the 24.4 kDa AA9 family lytic polysaccharide monooxygenase TaLPMO9A from Thermoascus aurantiacus has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1H, 13C, and 15N chemical shift assignments, as well as an analysis of the secondary structure of the protein based on the secondary chemical shifts.

    DOI: 10.1007/s12104-018-9839-y

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  • Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis. International journal

    Yiming Feng, Yoshihito Kitaoku, Jun Tanaka, Toki Taira, Takayuki Ohnuma, Finn L Aachmann, Tamo Fukamizo

    Plant molecular biology   97 ( 6 )   553 - 564   2018.8

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    KEY MESSAGE: Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds. However, chitin-mediated defense system intensively studied in higher plants remains to be investigated in this microalga. Recently, Taira et al. (Biosci Biotechnol Biochem 82:1090-1100, 2018) isolated a unique chitinase gene, comprising two catalytic domains almost homologous to each other (Cat1 and Cat2) and two chitin-binding domains (CBD1 and CBD2), from E. gracilis. We herein examined the mode of action and the specificity of the recombinant Cat2 by size exclusion chromatography and NMR spectroscopy. Both Cat1 and Cat2 appeared to act toward chitin substrate with non-processive/endo-splitting mode, recognizing two contiguous N-acetylglucosamine units at subsites - 2 and - 1. This is the first report on a chitinase having two endo-splitting catalytic domains. A cooperative action of two different endo-splitting domains may be advantageous for defensive action of the E. gracilis chitinase. The unicellular alga, E. gracilis, produces a chitinase consisting of two GH18 catalytic domains (Cat1 and Cat2) and two CBM18 chitin-binding domains (CBD1 and CBD2). Here, we produced a recombinant protein of the Cat2 domain to examine its mode of action as well as specificity. Cat2 hydrolyzed N-acetylglucosamine (A) oligomers (An, n = 4, 5, and 6) and partially N-acetylated chitosans with a non-processive/endo-splitting mode of action. NMR analysis of the product mixture from the enzymatic digestion of chitosan revealed that the reducing ends were exclusively A-unit, and the nearest neighbors of the reducing ends were mostly A-unit but not exclusively. Both A-unit and D-unit were found at the non-reducing ends and the nearest neighbors. These results indicated strong and absolute specificities for subsites - 2 and - 1, respectively, and no preference for A-unit at subsites + 1 and + 2. The same results were obtained from sugar sequence analysis of the individual enzymatic products from the chitosans. The subsite specificities of Cat2 are similar to those of GH18 human chitotriosidase, but differ from those of plant GH18 chitinases. Since the structures of Cat1 and Cat2 resemble to each other (99% similarity in amino acid sequences), Cat1 may hydrolyze the substrate with the same mode of action. Thus, the E. gracilis chitinase appears to act toward chitin polysaccharide chain through a cooperative action of the two endo-splitting catalytic domains, recognizing two contiguous A-units at subsites - 2 and - 1.

    DOI: 10.1007/s11103-018-0759-0

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  • Structure and function of a novel periplasmic chitooligosaccharide-binding protein from marine Vibrio bacteria. International journal

    Wipa Suginta, Natchanok Sritho, Araya Ranok, David Michael Bulmer, Yoshihito Kitaoku, Bert van den Berg, Tamo Fukamizo

    The Journal of biological chemistry   293 ( 14 )   5150 - 5159   2018.4

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    Periplasmic solute-binding proteins in bacteria are involved in the active transport of nutrients into the cytoplasm. In marine bacteria of the genus Vibrio, a chitooligosaccharide-binding protein (CBP) is thought to be the major solute-binding protein controlling the rate of chitin uptake in these bacteria. However, the molecular mechanism of the CBP involvement in chitin metabolism has not been elucidated. Here, we report the structure and function of a recombinant chitooligosaccharide-binding protein from Vibrio harveyi, namely VhCBP, expressed in Escherichia coli Isothermal titration calorimetry revealed that VhCBP strongly binds shorter chitooligosaccharides ((GlcNAc) n , where n = 2, 3, and 4) with affinities that are considerably greater than those for glycoside hydrolase family 18 and 19 chitinases but does not bind longer ones, including insoluble chitin polysaccharides. We also found that VhCBP comprises two domains with flexible linkers and that the domain-domain interface forms the sugar-binding cleft, which is not long extended but forms a small cavity. (GlcNAc)2 bound to this cavity, apparently triggering a closed conformation of VhCBP. Trp-363 and Trp-513, which stack against the two individual GlcNAc rings, likely make a major contribution to the high affinity of VhCBP for (GlcNAc)2 The strong chitobiose binding, followed by the conformational change of VhCBP, may facilitate its interaction with an active-transport system in the inner membrane of Vibrio species.

    DOI: 10.1074/jbc.RA117.001012

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  • Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis. International journal

    Takayuki Ohnuma, Toki Taira, Naoyuki Umemoto, Yoshihito Kitaoku, Morten Sørlie, Tomoyuki Numata, Tamo Fukamizo

    Biochemical and biophysical research communications   494 ( 3-4 )   736 - 741   2017.12

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    We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule. The free energy changes (ΔGr°) for binding of (GlcNAc)6, (GlcNAc)5, and (GlcNAc)4 to PrLysM2 were determined to be -5.4, -5,4 and -4.6 kcal mol-1, respectively, by ITC. Thermodynamic dissection of the binding energetics of (GlcNAc)6 revealed that the driving force is the enthalpy change (ΔHr° = -11.7 ± 0.2 kcal/mol) and the solvation entropy change (-TΔSsolv° = -5.9 ± 0.6 kcal/mol). This is the first description of thermodynamic signatures of a chitin oligosaccharide binding to a LysM module.

    DOI: 10.1016/j.bbrc.2017.08.143

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  • Chitin oligosaccharide binding to the lysin motif of a novel type of chitinase from the multicellular green alga, Volvox carteri. International journal

    Yoshihito Kitaoku, Tamo Fukamizo, Tomoyuki Numata, Takayuki Ohnuma

    Plant molecular biology   93 ( 1-2 )   97 - 108   2017.1

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    KEY MESSAGE: The chitinase-mediated defense system in higher plants has been intensively studied from physiological and structural viewpoints. However, the defense system in the most primitive plant species, such as green algae, has not yet been elucidated in details. In this study, we solved the crystal structure of a family CBM-50 LysM module attached to the N-terminus of chitinase from Volvox carteri, and successfully analyzed its chitin-binding ability by NMR spectroscopy and isothermal titration calorimetry. Trp96 of the LysM module appeared to make a CH-π stacking interaction with the reducing end sugar residue of the ligand. We believe the data included in this manuscript provide novel insights into the molecular basis of chitinase-mediated defense system in green algae. A chitinase from the multicellular green alga, Volvox carteri, contains two N-terminal lysin motifs (VcLysM1 and VcLysM2), that belong to the CBM-50 family, in addition to a catalytic domain. We produced a recombinant protein of VcLysM2 in order to examine its structure and function. The X-ray crystal structure of VcLysM2 was successfully solved at a resolution of 1.2 Å, and revealed that the protein adopts the βααβ fold typical of members belonging to the CBM-50 family. NMR spectra of 13C- and 15N-labeled proteins were analyzed in order to completely assign the main chain resonances of the 1H,15N-HSQC spectrum in a sequential manner. NMR-based titration experiments of chitin oligosaccharides, (GlcNAc)n (n = 3-6), revealed the ligand-binding site of VcLysM2, in which the Trp96 side chain appeared to interact with the terminal GlcNAc residue of the ligand. We then mutated Trp96 to alanine (VcLysM2-W96A), and the mutant protein was characterized. Based on isothermal titration calorimetry, the affinity of (GlcNAc)6 toward VcLysM2 (-6.9 kcal/mol) was found to be markedly higher than that of (GlcNAc)3 (-4.1 kcal/mol), whereas the difference in affinities between (GlcNAc)6 and (GlcNAc)3 in VcLysM2-W96A (-5.1 and -4.0 kcal/mol, respectively) was only moderate. This suggests that the Trp96 side chain of VcLysM2 interacts with the sugar residue of (GlcNAc)6 not with (GlcNAc)3. VcLysM2 appears to preferentially bind (GlcNAc)n with longer chains and plays a major role in the degradation of the chitinous components of enzyme targets.

    DOI: 10.1007/s11103-016-0549-5

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  • Mechanism of chitosan recognition by CBM32 carbohydrate-binding modules from a Paenibacillus sp. IK-5 chitosanase/glucanase. International journal

    Shoko Shinya, Shigenori Nishimura, Yoshihito Kitaoku, Tomoyuki Numata, Hisashi Kimoto, Hideo Kusaoke, Takayuki Ohnuma, Tamo Fukamizo

    The Biochemical journal   473 ( 8 )   1085 - 95   2016.4

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    An antifungal chitosanase/glucanase isolated from the soil bacterium Paenibacillus sp. IK-5 has two CBM32 chitosan-binding modules (DD1 and DD2) linked in tandem at the C-terminus. In order to obtain insights into the mechanism of chitosan recognition, the structures of DD1 and DD2 were solved by NMR spectroscopy and crystallography. DD1 and DD2 both adopted a β-sandwich fold with several loops in solution as well as in crystals. On the basis of chemical shift perturbations in(1)H-(15)N-HSQC resonances, the chitosan tetramer (GlcN)4 was found to bind to the loop region extruded from the core β-sandwich of DD1 and DD2. The binding site defined by NMR in solution was consistent with the crystal structure of DD2 in complex with (GlcN)3, in which the bound (GlcN)3 stood upright on its non-reducing end at the binding site. Glu(14)of DD2 appeared to make an electrostatic interaction with the amino group of the non-reducing end GlcN, and Arg(31), Tyr(36)and Glu(61)formed several hydrogen bonds predominantly with the non-reducing end GlcN. No interaction was detected with the reducing end GlcN. Since Tyr(36)of DD2 is replaced by glutamic acid in DD1, the mutation of Tyr(36)to glutamic acid was conducted in DD2 (DD2-Y36E), and the reverse mutation was conducted in DD1 (DD1-E36Y). Ligand-binding experiments using the mutant proteins revealed that this substitution of the 36th amino acid differentiates the binding properties of DD1 and DD2, probably enhancing total affinity of the chitosanase/glucanase toward the fungal cell wall.

    DOI: 10.1042/BCJ20160045

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  • A class III chitinase without disulfide bonds from the fern, Pteris ryukyuensis: crystal structure and ligand-binding studies. International journal

    Yoshihito Kitaoku, Naoyuki Umemoto, Takayuki Ohnuma, Tomoyuki Numata, Toki Taira, Shohei Sakuda, Tamo Fukamizo

    Planta   242 ( 4 )   895 - 907   2015.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    MAIN CONCLUSION: We first solved the crystal structure of class III catalytic domain of a chitinase from fern (PrChiA-cat), and found a structural difference between PrChiA-cat and hevamine. PrChiA-cat was found to have reduced affinities to chitin oligosaccharides and allosamidin. Plant class III chitinases are subdivided into enzymes with three disulfide bonds and those without disulfide bonds. We here referred to the former enzymes as class IIIa chitinases and the latter as class IIIb chitinases. In this study, we solved the crystal structure of the class IIIb catalytic domain of a chitinase from the fern Pteris ryukyuensis (PrChiA-cat), and compared it with that of hevamine, a class IIIa chitinase from Hevea brasiliensis. PrChiA-cat was found to adopt an (α/β)8 fold typical of GH18 chitinases in a similar manner to that of hevamine. However, PrChiA-cat also had two large loops that extruded from the catalytic site, and the corresponding loops in hevamine were markedly smaller than those of PrChiA-cat. An HPLC analysis of the enzymatic products revealed that the mode of action of PrChiA-cat toward chitin oligosaccharides, (GlcNAc) n (n = 4-6), differed from those of hevamine and the other class IIIa chitinases. The binding affinities of (GlcNAc)3 and (GlcNAc)4 toward the inactive mutant of PrChiA-cat were determined by isothermal titration calorimetry, and were markedly lower than those toward other members of the GH18 family. The affinity and the inhibitory activity of allosamidin toward PrChiA-cat were also lower than those toward the GH18 chitinases investigated to date. Several hydrogen bonds found in the crystal structure of hevamine-allosamidin complex were missing in the modeled structure of PrChiA-cat-allosamidin complex. The structural findings for PrChiA-cat successfully interpreted the functional data presented.

    DOI: 10.1007/s00425-015-2330-4

    PubMed

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MISC

Awards

  • Excellent Poster Award

    2014.11   MIE - BIOFORUM 2014  

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Research Projects

  • NMR analysis of the structure and function of chitin-active enzymes

    2017.05 - 2017.11

    9 th Kyoto International Forum for Environment and Energy  9 th Kyoto International Forum for Environment and Energy Mobility grant 

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  • LysM型糖質結合モジュールの構造生物学ー植物-微生物間相互作用の制御を目指して

    Grant number:16J10483  2016.04 - 2018.03

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    北奥 喜仁

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    Grant amount:\1300000 ( Direct expense: \1300000 )

    植物-微生物間相互作用において、LysM型糖質結合モジュール(以下、LysM)は、植物と微生物の双方が産生するタンパク質中の構成ドメインとして存在しており、微生物の細胞壁成分、キチンやペプチドグリカンを媒介とした相互作用のインターフェースで機能している。本研究では、植物と微生物がもつLysMの機能を調べることによって、植物-微生物間相互作用の分子基盤を明らかにすることを目的としている。前年度に引き続き、ボルボックス由来のキチナーゼ(VcChi)がもつLysMを大腸菌発現系で発現させ、LysMがタンデム構造をとることによるリガンド結合への影響を調べた。リガンドとして、微生物の細胞壁成分に類似した構造をもち、さらに詳細な性質が明らかにされているキチンオリゴ糖を用いて実験を行った。その結果、キチンオリゴ糖に対する結合力は、タンデム構造を形成することで、低下することがわかった。このことから、VcChiは長鎖の基質の分解を触媒する酵素であることが示唆された。
    2つのLysMをもつ真菌由来のエフェクタータンパク質の発現系を構築することとした。幾つかのタグタンパク質を付加した状態で発現条件を検討したところ、チオレドキシンタグを付加した目的タンパク質の大腸菌での発現を確認した。糖鎖修飾が安定性に影響するとの報告を考慮し、リガンド存在下で精製することで、単一のタンパク質を調製することができた。今後、このタンパク質を用いて、構造と機能の解析を行い、植物キチナーゼがもつLysMと比較検討する予定である。

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  • Mode of action of plant GH18 chitinases toward chitosan

    Scandinavia-Japan Sasakawa Foundation 

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