Applied Microbiology and Biotechnology

, Volume 100, Issue 24, pp 10403–10415 | Cite as

X-ray structures of the Pseudomonas cichorii D-tagatose 3-epimerase mutant form C66S recognizing deoxy sugars as substrates

  • Hiromi Yoshida
  • Akihide Yoshihara
  • Tomohiko Ishii
  • Ken Izumori
  • Shigehiro KamitoriEmail author
Biotechnologically relevant enzymes and proteins


Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.


β/α-Barrel Deoxy sugar Epimerase Rare sugar X-ray structure 



We thank Mr. Y. Tahara, Ms. S. Kayahara, and Mr. S. Ohga for assisting with protein purification; Mr. K. Yube for his technical assistance with DNA sequencing; and Dr. K. Inaka, Dr. N. Furubayashi, Dr. M. Yamada, and Dr. K. Ohta for assisting with crystallizations under microgravity. This research was performed with the approval of the Photon Factory Advisory Committee and National Laboratory for High Energy Physics, Japan. This study is contributed by a part of “High-Quality Protein Crystal Growth Experiment on KIBO” promoted by JAXA (Japan Aerospace Exploration Agency). Russian spacecraft “Progress” and/or “Soyuz” provided by the Russian Federal Space Agency were used for space transportation. A part of space crystallization technology had been developed by the European Space Agency and University of Granada.

Compliance with ethical standards


This study was funded in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (25440028, 23770122).

Conflict interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

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Fig. S1 (PDF 218 KB)
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Fig. S4 (PDF 458 KB)


  1. Brunger AT (1993) X-PLOR 3.1: a system for X-ray crystallography and NMR. Yale University Press, New Haven and LondonGoogle Scholar
  2. Carrell HL, Glusker JP, Burger V, Manfre F, Tritsch D, Biellmann JF (1989) X-ray analysis of D-xylose isomerase at 1.9 A: native enzyme in complex with substrate and with a mechanism-designed inactivator. Proc Natl AcadSci USA 86:4440–4444CrossRefGoogle Scholar
  3. Carrell HL, Hoier H, Glusker JP (1994) Modes of binding substrates and their analogues to the enzyme D-xylose isomerase. Acta Crystallogr Sect D 50:113–123. doi: 10.1107/S0907444993009345 CrossRefGoogle Scholar
  4. Collyer CA, Henrick K, Blow DM (1990) Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift. J Mol Biol 212:211–235. doi: 10.1016/0022-2836(90)90316-E CrossRefPubMedGoogle Scholar
  5. Emsley P, Lohkamp B, Scott W, Cowtan K (2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66:486–501. doi: 10.1107/S0907444910007493 CrossRefPubMedPubMedCentralGoogle Scholar
  6. Fenn TD, Ringe D, Petsko GA (2004) Xylose isomerase in substrate and inhibitor michaelis states: atomic resolution studies of a metal-mediated hydride shift. Biochemistry 43:6464–6474. doi: 10.1021/bi049812o CrossRefPubMedGoogle Scholar
  7. Gullapalli P, Yoshihara A, Morimoto K, Rao D, Akimitsu K, Jenkinson SF, Fleet GWJ, Izumori K (2010) Conversion of l-rhamnose into ten of the sixteen 1- and 6-deoxyketohexoses in water with three reagents: d-tagatose-3-epimerase equilibrates C3 epimers of deoxyketoses. Tetrahedron Lett 51(6):895–898. doi: 10.1016/j.tetlet.2009.12.024 CrossRefGoogle Scholar
  8. Hayashi N, Iida T, Yamada T, Okuma K, Takehara I, Yamamoto T, Yamada K, Tokuda M (2010) Study on the postprandial blood glucose suppression effect of D-psicose in borderline diabetes and the safety of long-term ingestion by normal human subjects. Biosci Biotechnol Biochem 74:510–519. doi: 10.1271/bbb.90707 CrossRefPubMedGoogle Scholar
  9. Hayashi N, Yamada T, Takamine S, Iida T, Okuma K, Tokuda M (2014) Weight reducing effect and safety evaluation of rare sugar syrup by a randomized double-blind, parallel-group study in human. J Funct Foods 11:152–159. doi: 10.1016/j.jff.2014.09.020 CrossRefGoogle Scholar
  10. Hossain A, Yamaguchi F, Matsunaga T, Hirata Y, Kamitori K, Dong Y, Sui L, Tsukamoto I, Ueno M, Tokuda M (2012) Rare sugar D-psicose protects pancreas β-islets and thus improves insulin resistance in OLETF rats. Biochem Biophys Res Commun 425:717–723. doi: 10.1016/j.bbrc.2012.07.135 CrossRefPubMedGoogle Scholar
  11. Hossain MA, Kitagaki S, N akano D, Nishiyama A, Funamoto Y, Matsunaga T, Tsukamoto I, Yamaguchi F, Kamitori K, Dong Y, Hirata Y, Murao K, Toyoda Y, Tokuda M (2011) Rare sugar D-psicose improves insulin sensitivity and glucose tolerance in type 2 diabetes Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Biochem Biophys Res Commun 405:7–12. doi: 10.1016/j.bbrc.2010.12.091 CrossRefPubMedGoogle Scholar
  12. Iida T, Kishimoto Y, Yoshikawa Y, Hayashi N, Okuma K, Tohi M, Yagi K, Matsuo T, Izumori K (2008) Acute D-psicose administration decreases the glycemic responses to an oral maltodextrin tolerance test in normal adults. J Nutr Sci Vitaminol (Tokyo) 54:511–514. doi: 10.3177/jnsv.54.511 CrossRefGoogle Scholar
  13. Iida T, Yamada T, Hayashi N, Okuma K, Izumori K, Ishii R, Matsuo T (2013) Reduction of abdominal fat accumulation in rats by 8-week ingestion of a newly developed sweetener made from high fructose corn syrup. Food Chem 138(2–3):781–785. doi: 10.1016/j.foodchem.2012.11.017 CrossRefPubMedGoogle Scholar
  14. Ishida Y, Kamiya T, Izumori K (1997) Production of d-tagatose 3-epimerase of Pseudomonas cichorii ST-24 using recombinant Escherichia coli. J Ferment Bioeng 84:348–350. doi: 10.1016/S0922-338X(97)89257-4 CrossRefGoogle Scholar
  15. Itoh H, Okaya H, Khan AR, Tajima S, Hayakawa S, Izumori K (1994) Purification and characterization of D-tagatose 3-epimerase from Pseudomonas sp. ST-24. Biosci Biotechnol Biochem 58:2168–2171. doi: 10.1271/bbb.58.2168 CrossRefGoogle Scholar
  16. Itoh H, Sato T, Izumori K (1995) Preparation of D-psicose from D-fructose by immobilized D-tagatose 3-epimerase. J Ferment Bioeng 80:101–103. doi: 10.1016/0922-338X(95)98186-O CrossRefGoogle Scholar
  17. Izumori K, Khan AR, Okaya H, Tsumura T (1993) A new enzyme, D-ketohexose 3-epimerase, from Pseudomonas sp. ST-24. Biosci Biotechnol Biochem 57:1037–1039. doi: 10.1271/bbb.57.1037 CrossRefGoogle Scholar
  18. Jia M, Mu WM, Chu FF, Zhang XM, Jiang B, Zhou LL, Zhang T (2014) A D-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for D-psicose production: cloning, expression, purification, and characterization. Appl Microbiol Biotechnol 98:717–725. doi: 10.1007/s00253-013-4924-8 CrossRefPubMedGoogle Scholar
  19. Kim HJ, Hyun EK, Kim YS, Lee YJ, Oh DK (2006a) Characterization of an Agrobacterium tumefaciens D-psicose 3-epimerase that converts D-fructose to D-psicose. Appl Environ Microbiol 72:981–985. doi: 10.1128/AEM.72.2.981-985.2006 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kim K, Kim HJ, Oh DK, Cha SS, Rhee S (2006b) Crystal structure of D-psicose 3-epimerase from Agrobacterium tumefaciens and its complex with true substrate D-fructose: a pivotal role of metal in catalysis, an active site for the non-phosphorylated substrate, and its conformational changes. J Mol Biol 361:920–931. doi: 10.1016/j.jmb.2006.06.069 CrossRefPubMedGoogle Scholar
  21. Kovalevsky AY, Hanson L, Fisher SZ, Mustyakimov M, Mason SA, Forsyth VT, Blakeley MP, Keen DA, Wagner T, Carrell HL, Katz AK, Glusker JP, Langan P (2010) Metal ion roles and the movement of hydrogen during reaction catalyzed by D-xylose isomerase: a joint x-ray and neutron diffraction study. Structure 18:688–699. doi: 10.1016/j.str.2010.03.011 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372:774–797. doi: 10.1016/j.jmb.2007.05.022 CrossRefPubMedGoogle Scholar
  23. Langan P, Sangha AK, Wymore T, Parks JM, Yang ZK, Hanson BL, Fisher Z, Mason SA, Blakeley MP, Forsyth VT, Glusker JP, Carrell HL, Smith JC, Keen DA, Graham DE, Kovalevsky A (2014) L-Arabinose binding, isomerization, and epimerization by D-xylose isomerase: X-ray/neutron crystallographic and molecular simulation study. Structure 22:1287–1300. doi: 10.1016/j.str.2014.07.002 CrossRefPubMedGoogle Scholar
  24. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1992) PROCHECK v.2: programs to check the stereochemical quality of protein structures. Oxford Molecular Ltd, Oxford, EnglandGoogle Scholar
  25. Matsuo T, Izumori K (2009) D-Psicose inhibits intestinal alpha glucosidase and suppresses the glycemic response after ingestion of carbohydrates in rats. J Clin Biochem Nutr 45:202–206. doi: 10.3164/jcbn.09-36 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Matsuo T, Suzuki H, Hashiguchi M, Izumori K (2002) D-Psicose is a rare sugar that provides no energy to growing rats. J Nutr Sci Vitaminol 48:77–80. doi: 10.3177/jnsv.48.77 CrossRefPubMedGoogle Scholar
  27. Matsuo T (2006) Inhibitory effects of D-psicose on glycemic responses after oral carbohydrate tolerance test in rats. J Jpn Soc Nutr Food Sci 59:119–121. doi: 10.4327/jsnfs.59.119 CrossRefGoogle Scholar
  28. McRee DE (1999) XtalView/Xfit: a versatile program for manipulating atomic coordinate and electron density. J Struct Biol 125:156–165. doi: 10.1006/jsbi.1999.4094 CrossRefPubMedGoogle Scholar
  29. Mu W, Zhang W, Feng Y, Jiang B, Zhou L (2012) Recent advances on applications and biotechnological production of D-psicose. Appl Microbiol Biotechnol 94:1461–1467. doi: 10.1007/s00253-012-4093-1 CrossRefPubMedGoogle Scholar
  30. Mu WM, Chu FF, Xing QC, Yu SH, Zhou L, Jiang B (2011) Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10. J Agric Food Chem 59:7785–7792. doi: 10.1021/jf201356q CrossRefPubMedGoogle Scholar
  31. Mu WM, Zhang WL, Fang D, Zhou L, Jiang B, Zhang T (2013) Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from Clostridium sp. Biotechnol Lett 35:1481–1486. doi: 10.1007/s10529-013-1230-6 CrossRefPubMedGoogle Scholar
  32. Munshi P, Snell EH, van der Woerd MJ, Judge RA, Myles DA, Ren Z, Meilleur F (2014) Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant. Acta Crystallogr D Biol Crystallogr 70:414–420. doi: 10.1107/S1399004713029684 CrossRefPubMedGoogle Scholar
  33. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53:240–255. doi: 10.1107/S0907444996012255 CrossRefPubMedGoogle Scholar
  34. Nakajima Y, Gotanda T, Uchiyama H, Furukawa T, Haraguchi M, Ikeda R, Sumizawa T, Yoshida H, Akiyama S (2004) Inhibition of metastasis of tumor cells overexpressing thymidine phosphorylase by 2-deoxy-L-ribose. Cancer Res 64:1794–1801CrossRefPubMedGoogle Scholar
  35. Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Method in enzymology 276: macromolecular crystallography, part A, 307–326. doi:10.1016/S0076–6879(97)76066-XGoogle Scholar
  36. Shompoosang S, Yoshihara A, Uechi K, Asada Y, Morimoto K (2014) Enzymatic production of three 6-deoxy-aldohexoses from L-rhamnose. Biosci Biotechnol Biochem 78:317–325. doi: 10.1080/09168451.2014.878217 CrossRefPubMedGoogle Scholar
  37. Shompoosang S, Yoshihara A, Uechi K, Asada Y, Morimoto K (2016) Novel process for producing 6-deoxy monosaccharides from l-fucose by coupling and sequential enzymatic method. J Biosci Bioeng 121:1–6. doi: 10.1016/j.jbiosc.2015.04.017 CrossRefPubMedGoogle Scholar
  38. Takahashi S, Ohta K, Furubayashi N, Yan B, Koga M, Wada Y, Yamada M, Inaka K, Tanaka H, Miyoshi H, Kobayashi T, Kamigaichi S (2013) JAXA protein crystallization in space: ongoing improvements for growing high-quality crystals. J Synchrotron Radiat 20(Pt 6):968–973. doi: 10.1107/S0909049513021596 CrossRefPubMedPubMedCentralGoogle Scholar
  39. Terami Y, Yoshida H, Uechi K, Morimoto K, Takata G, Kamitori S (2015) Essentiality of tetramer formation of Cellulomonas parahominis L-ribose isomerase involved in novel L-ribose metabolic pathway. Appl Microbiol Biotechnol 99:6303–6313. doi: 10.1007/s00253-015-6417-4 CrossRefPubMedGoogle Scholar
  40. Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025. doi: 10.1107/S0021889897006766 CrossRefGoogle Scholar
  41. Whitlow M, Howard AJ, Finzel BC, Poulos TL, Winborne E, Gilliland GL (1991) A metal-mediated hydride shift mechanism for xylose isomerase based on the 1.6 Å Streptomyces rubiginosus structures with xylitol and D-xylose. Proteins 9:153–173. doi: 10.1002/prot.340090302 CrossRefPubMedGoogle Scholar
  42. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr 67:235–242. doi: 10.1107/S0907444910045749 CrossRefPubMedPubMedCentralGoogle Scholar
  43. Yoshida H, Yamada M, Nishitani T, Takada G, Izumori K, Kamitori S (2007) Crystal structures of D-tagatose 3-epimerase from Pseudomonas cichorii and its complexes with D-tagatose and D-fructose. J Mol Biol 374(2):443–453. doi: 10.1016/j.jmb.2007.09.033 CrossRefPubMedGoogle Scholar
  44. Yoshida H, Yamaji M, Ishii T, Izumori K, Kamitori S (2010) Catalytic reaction mechanism of Pseudomonas stutzeri L-rhamnose isomerase deduced from X-ray structures. FEBS J 277:1045–1057. doi: 10.1111/j.1742-4658.2009.07548.x CrossRefPubMedGoogle Scholar
  45. Yoshida H, Yoshihara A, Teraoka M, Terami Y, Takata G, Izumori K, Kamitori S (2014) X-ray structure of a novel L-ribose isomerase acting on a non-natural sugar L-ribose as its ideal substrate. FEBS J 281:3150–3164. doi: 10.1111/febs.12850 CrossRefPubMedGoogle Scholar
  46. Yoshida H, Yoshihara A, Teraoka M, Yamashita S, Izumori K, Kamitori S (2012) Structure of L-rhamnose isomerase in complex with L-rhamnopyranose demonstrates the sugar-ring opening mechanism and the role of a substrate sub-binding site. FEBS Open Bio 3:35–40. doi: 10.1016/j.fob.2012.11.008 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Zhang L, Mu W, Jiang B, Zhang T (2009) Characterization of D-tagatose-3-epimerase from Rhodobacter sphaeroides that converts D-fructose into D-psicose. Biotechnol Lett 31:857–862. doi: 10.1007/s10529-009-9942-3 CrossRefPubMedGoogle Scholar
  48. Zhang W, Fang D, Xing Q, Zhou L, Jiang B, Mu WM (2013b) Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704. PLoS One 8:e62987. doi: 10.1371/journal.pone.0062987 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Zhang W, Fang D, Zhang T, Zhou L, Jiang B, Mu W (2013a) Characterization of a metal-dependent D-psicose 3-epimerase from a novel strain, Desmospora sp. 8437. J Agric Food Chem 61:11468–11476. doi: 10.1021/jf4035817 CrossRefPubMedGoogle Scholar
  50. Zhang W, Zhang T, Jiang B, Mu W (2015) Biochemical characterization of a d-psicose 3-epimerase from Treponema primitia ZAS-1 and its application on enzymatic production of d-psicose. J Sci Food Agric 96:49–56. doi: 10.1002/jsfa.7187 CrossRefPubMedGoogle Scholar
  51. Zhu YM, Men Y, Bai W, Li XB, Zhang LL, Sun YX, Ma Y (2012) Overexpression of D-psicose 3-epimerase from Ruminococcus sp. in Escherichia coli and its potential application in D-psicose production. Biotechnol Lett 34:1901–1906. doi: 10.1007/s10529-012-0986-4 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Life Science Research Center and Faculty of MedicineKagawa UniversityKita-gunJapan
  2. 2.Rare Sugar Research Center and Faculty of AgricultureKagawa UniversityKita-gunJapan
  3. 3.Faculty of EngineeringKagawa UniversityTakamatsuJapan

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