Journal of Microbiology and Biotechnology
The Korean Society for Microbiology and Biotechnology publishes the Journal of Microbiology and Biotechnology.

2019 ; Vol.29-2: 244~255

AuthorSun-Ha Park, Sunghark Kwon, Chang Woo Lee, Chang Min Kim, Chang Sook Jeong, Kyung-Jin Kim, Jong Wook Hong, Hak Jun Kim, Hyun Ho Park, Jun Hyuck Lee
Place of dutyUnit of Polar Genomics, Korea Polar Research Institute, Republic of Korea
TitleCrystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.
PublicationInfo J. Microbiol. Biotechnol.2019 ; Vol.29-2
AbstractXylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of PbXI at 1.94-Å resolution to investigate the origin of its thermostability. The PbXI structure shows a (β/α)8-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the β4-α6 loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the β1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible β1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.
Full-Text
Key_wordCold-active protein, crystal structure, paenibacillus species, xylose isomerase, X-ray crystallography
References
  1. Mosier N, Wyman C, Dale B, Elander R, Lee Y, Holtzapple M, et al. 2005. Features of promising technologies for pretreatment of lignocellulosic biomass. Biores. Technol. 96: 673-686.
    Pubmed CrossRef
  2. Chiang L-C, Gong C-S, Chen L-F, Tsao GT. 1981. D-Xylulose fermentation to ethanol by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 42: 284-289.
    Pubmed Pubmed Central
  3. Chandrakant P, Bisaria V. 2000. Simultaneous bioconversion of glucose and xylose to ethanol by Saccharomyces cerevisiae in the presence of xylose isomerase. Appl. Microbiol. Biotechnol. 53: 301-309.
    Pubmed CrossRef
  4. Gong C-S, Chen L-F, Flickinger MC, Chiang L-C, Tsao GT. 1981. Production of ethanol from D-xylose by using D-xylose isomerase and yeasts. Appl. Environ. Microbiol. 41: 430-436.
    Pubmed Pubmed Central
  5. Zhou H, Cheng J-s, Wang BL, Fink GR, Stephanopoulos G. 2012. Xylose isomerase overexpression along with engineering of the pentose phosphate pathway and evolutionary engineering enable rapid xylose utilization and ethanol production by Saccharomyces cerevisiae. Metabol. Eng. 14: 6 11622.
  6. Madhavan A, Tamalampudi S, Ushida K, Kanai D, Katahira S, Srivastava A, et al. 2009. Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol. Appl. Microbiol. Biotechnol. 82: 1067-1068.
    Pubmed CrossRef
  7. Moes C J, Pretorius IS, v an Zyl W H. 1 996 . Cloning and expression of the Clostridium thermosulfurogenes D-xylose isomerase gene (xylA) in Saccharomyces cerevisiae. Biotechnol. Lett. 18: 269-274.
    CrossRef
  8. Sarthy A, McConaughy B, Lobo Z, Sundstrom J, Furlong C, Hall B. 1987. Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 53: 1996-2000.
    Pubmed Pubmed Central
  9. Kristo P, Saarelainen R, Fagerström R, Aho S, Korhola M. 1996. Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley. Eur. J. Biochem. 237: 240-246.
    Pubmed CrossRef
  10. Umemoto Y, Shibata T, Araki T. 2012. D-xylose isomerase from a marine bacterium, Vibrio sp. strain XY-214, and D-xylulose production from β-1, 3-xylan. Marine Biotechnol. 14: 10-20.
    CrossRef
  11. Son H, Lee S-M, Kim K-J. 2018. Crystal structure and biochemical characterization of xylose isomerase from Piromyces sp. E2. J. Microbiol. Biotechnol. 28: 571-578.
    Pubmed
  12. Dekker K, Yamagata H, Sakaguchi K, Udaka S. 1991. Xylose (glucose) isomerase gene from the thermophile Thermus thermophilus: cloning, sequencing, and comparison with other thermostable xylose isomerases. J. Bacteriol. 173: 3078-3083.
    Pubmed CrossRef Pubmed Central
  13. Park J-H, Batt CA. 2004. Restoration of a defective Lactococcus lactis xylose isomerase. Appl. Environ. Microbiol. 70: 4318-4325.
    Pubmed CrossRef Pubmed Central
  14. Kovalevsky AY, Hanson L, Fisher SZ, Mustyakimov M, Mason SA, Forsyth VT, et al. 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.
    Pubmed CrossRef Pubmed Central
  15. Chang C, Park BC, Lee D-S, Suh SW. 1999. Crystal structures of thermostable xylose isomerases from Thermus caldophilus and Thermus thermophilus: possible structural determinants of thermostability. J. Mol. Biol. 288: 623-634.
    Pubmed CrossRef
  16. Allen KN, Lavie A, Glasfeld A, Tanada TN, Gerrity DP, Carlson SC, et al. 1994. Role of the divalent metal ion in sugar binding, ring opening, and isomerization by D-xylose isomerase: replacement of a catalytic metal by an amino acid. Biochemistry 33: 1488-1494.
    Pubmed CrossRef
  17. Bae J-E, Hwang KY, Nam KH. 2018. Structural analysis of substrate recognition by glucose isomerase in Mn2+ binding mode at M2 site in S. rubiginosus. Biochem. Biophys. Res. Commun. 503: 770-775.
    Pubmed CrossRef
  18. Toteva MM, Silvaggi NR, Allen KN, Richard JP. 2011. Binding energy and catalysis by D-xylose isomerase: kinetic, product, and X-ray crystallographic analysis of enzyme catalyzed isomerization of (R)-glyceraldehyde. Biochemistry 50: 10170-10181.
    Pubmed CrossRef Pubmed Central
  19. Carrell H, Rubin BH, Hurley TJ, Glusker JP. 1984. X-ray crystal structure of D-xylose isomerase at 4-A resolution. J. Biol. Chem. 259: 3230-3236.
    Pubmed
  20. Jenkins J, Janin J, Rey F, Chiadmi M, Van Tilbeurgh H, Lasters I, et al. 1992. Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites. Biochemistry 31: 5449-5458.
    Pubmed CrossRef
  21. Han B, Bong SM, Cho J, Kim M, Kim SJ, Lee BI. 2015. Crystal structure of a Class 2 D-xylose isomerase from the human intestinal tract microbe Bacteroides thetaiotaomicron. Biodesign 289: 41-47.
  22. Lee M, Rozeboom HtJ, de Waal PP, de Jong RM, Dudek HM, Janssen DB. 2017. Metal dependence of the xylose isomerase from Piromyces sp. E2 explored by activity profiling and protein crystallography. Biochemistry 56: 5991-6005.
    Pubmed CrossRef Pubmed Central
  23. Lee C, Bagdasarian M, Meng M, Zeikus J. 1990. Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine. J. Biol. Chem. 265:19082-19090.
    Pubmed
  24. Isaksen GV, Åqvist J, Brandsdal BO. 2014. Protein surface softness is the origin of enzyme cold-adaptation of trypsin. PLoS Comput. Biol. 10: e1003813.
    Pubmed CrossRef Pubmed Central
  25. Papaleo E, Pasi M, Riccardi L, Sambi I, Fantucci P, Gioia LD. 2008. Protein flexibility in psychrophilic and mesophilic trypsins. Evidence of evolutionary conservation of protein dynamics in trypsin-like serine-proteases. FEBS Lett. 582:1008-1018.
    Pubmed CrossRef
  26. Santiago M, Ramírez-Sarmiento CA, Zamora RA, Parra LP. 2016. Discovery, molecular mechanisms, and industrial applications of cold-active enzymes. Front. Microbiol. 7: 1408.
    Pubmed CrossRef Pubmed Central
  27. Cavicchioli R, Siddiqui KS, Andrews D, Sowers KR. 2002. Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13: 253-261.
    CrossRef
  28. Joseph B, Ramteke PW, Thomas G, Shrivastava N. 2007. Cold-active microbial lipases: a versatile tool for industrial applications. Biotechnol. Mol. Biol. Rev. 2: 39-48.
  29. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ, Disz T, et al. 2013. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res. 42: D206-D214.
    Pubmed CrossRef Pubmed Central
  30. Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:307-326.
    CrossRef
  31. Vagin A, Teplyakov A. 1997. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30:1022-1025.
    CrossRef
  32. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, et al. 2011. Overview of the CCP4 suite and current developments. Acta Crystallographica Section D:Biological Crystallography 67: 235-242.
    Pubmed CrossRef Pubmed Central
  33. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D, Biol. Crystallogr. 60:2126-2132.
    Pubmed CrossRef
  34. Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, et al. 2011. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D, Biol. Crystallogr. 67: 355-367.
    Pubmed CrossRef Pubmed Central
  35. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. 2010. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D, Biol. Crystallogr. 66: 12-21.
    Pubmed CrossRef Pubmed Central
  36. Holm L, Rosenström P. 2010. Dali server: conservation mapping in 3D. Nucleic Acids Res. 38: W545-W549.
    Pubmed CrossRef Pubmed Central
  37. Vieille C, Epting KL, Kelly RM, Zeikus JG. 2001. Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase. Eur. J. Biochem. 268: 6291-6301.
    Pubmed CrossRef
  38. Sriprapundh D, Vieille C, Zeikus JG. 2000. Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. Protein Eng. 13: 259-265.
    Pubmed CrossRef
  39. Vieille C, Hess JM, Kelly RM, Zeikus JG. 1995. xylA cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl. Environ. Microbiol. 61: 1867-1875.
    Pubmed Pubmed Central
  40. Sugiyama S, Maruyama M, Sazaki G, Hirose M, Adachi H, Takano K, et al. 2012. Growth of protein crystals in hydrogels prevents osmotic shock. J. Am. Chem. Soc. 134:5786-5789.
    Pubmed CrossRef
  41. Lavie A, Allen KN, Petsko GA, Ringe D. 1994. X-ray crystallographic structures of D-xylose isomerase-substrate complexes position the substrate and provide evidence for metal movement during catalysis. Biochemistry 33: 5469-5480.
    Pubmed CrossRef
  42. Zhu X-Y, Teng Mk, Niu L-W, Xu C, Wang Y-Z. 2000. Structure of xylose isomerase from Streptomyces diastaticus No. 7 strain M1033 at 1.85 Å resolution. Acta Crystallogr. D, Biol. Crystallogr. 56: 129-136.
    Pubmed CrossRef



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