Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Crystal Structure and Molecular Mechanism of Phosphotransbutyrylase from Clostridium acetobutylicum

  • Kim, Sangwoo (School of Life Sciences, BK21 FOUR KNU Creative BioSesearch Group, Kyungpook National University) ;
  • Kim, Kyung-Jin (School of Life Sciences, BK21 FOUR KNU Creative BioSesearch Group, Kyungpook National University)
  • Received : 2021.09.16
  • Accepted : 2021.09.23
  • Published : 2021.10.28
Download PDF
⟨ Previous Next ⟩

Abstract

Acetone-butanol-ethanol (ABE) fermentation by the anaerobic bacterium Clostridium acetobutylicum has been considered a promising process of industrial biofuel production. Phosphotransbutyrylase (phosphate butyryltransferase, PTB) plays a crucial role in butyrate metabolism by catalyzing the reversible conversion of butyryl-CoA into butyryl phosphate. Here, we report the crystal structure of PTB from the Clostridial host for ABE fermentation, C. acetobutylicum, (CaPTB) at a 2.9 Å resolution. The overall structure of the CaPTB monomer is quite similar to those of other acyltransferases, with some regional structural differences. The monomeric structure of CaPTB consists of two distinct domains, the N- and C-terminal domains. The active site cleft was formed at the interface between the two domains. Interestingly, the crystal structure of CaPTB contained eight molecules per asymmetric unit, forming an octamer, and the size-exclusion chromatography experiment also suggested that the enzyme exists as an octamer in solution. The structural analysis of CaPTB identifies the substrate binding mode of the enzyme and comparisons with other acyltransferase structures lead us to speculate that the enzyme undergoes a conformational change upon binding of its substrate.

Keywords

Acknowledgement

This research was supported by Kyungpook National University Development Project Research Fund, 2018.

References

  1. Jones DT, Woods DR. 1986. Acetone-butanol fermentation revisited. Microbiol. Rev. 50: 484-524. https://doi.org/10.1128/mr.50.4.484-524.1986
  2. Matta-el-Ammouri G, Janati-Idrissi R, Junelles AM, Petitdemange H, Gay R. 1987. Effects of butyric and acetic acids on acetone-butanol formation by Clostridium acetobutylicum. Biochimie 69: 109-115. https://doi.org/10.1016/0300-9084(87)90242-2
  3. Amador-Noguez D, Brasg IA, Feng X-J, Roquet N, Rabinowitz JD. 2011. Metabolome remodeling during the acidogenic-solventogenic transition in Clostridium acetobutylicum. Appl. Environ. Microbiol. 77: 7984-7997. https://doi.org/10.1128/AEM.05374-11
  4. Speakman HB. 1920. Gas production during the acetone and butyl alcohol fermentation of starch. J. Biol. Chem. 43: 401-411. https://doi.org/10.1016/S0021-9258(18)86291-3
  5. Millat T, Janssen H, Thorn GJ, King JR, Bahl H, Fischer R-J, et al. 2013. A shift in the dominant phenotype governs the pH-induced metabolic switch of Clostridium acetobutylicumin phosphate-limited continuous cultures. Appl. Microbiol. Biotechnol. 97: 6451-6466. https://doi.org/10.1007/s00253-013-4860-7
  6. Millat T, Janssen H, Bahl H, Fischer RJ, Wolkenhauer O. 2013. Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum in continuous culture. Microb. Biotechnol. 6: 526-539. https://doi.org/10.1111/1751-7915.12033
  7. Jones SW, Paredes CJ, Tracy B, Cheng N, Sillers R, Senger RS, et al. 2008. The transcriptional program underlying the physiology of clostridial sporulation. Genome Biol. 9: R114. https://doi.org/10.1186/gb-2008-9-7-r114
  8. Janssen H, Doring C, Ehrenreich A, Voigt B, Hecker M, Bahl H, et al. 2010. A proteomic and transcriptional view of acidogenic and solventogenic steady-state cells of Clostridium acetobutylicum in a chemostat culture. Appl. Microbiol. Biotechnol. 87: 2209-2226. https://doi.org/10.1007/s00253-010-2741-x
  9. Grimmler C, Janssen H, Kraube D, Fischer R-J, Bahl H, Durre P, et al. 2011. Genome-wide gene expression analysis of the switch between acidogenesis and solventogenesis in continuous cultures of Clostridium acetobutylicum. J. Mol. Microbiol. Biotechnol. 20: 1-15. https://doi.org/10.1159/000320973
  10. Cary JW, Petersen DJ, Papoutsakis ET, Bennett GN. 1988. Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli. J. Bacteriol. 170: 4613-4618. https://doi.org/10.1128/jb.170.10.4613-4618.1988
  11. Papoutsakis E, Bennett G. 1997. Molecular regulation and metabolic engineering of solvent production by Clostridium acetobutylicum. Bioprocess Technol. 24: 253-280.
  12. Steinbuchel A, Lutke-Eversloh T. 2003. Metabolic engineering and pathway construction for biotechnological production of relevant polyhydroxyalkanoates in microorganisms. Biochem. Eng. J. 16: 81-96. https://doi.org/10.1016/S1369-703X(03)00036-6
  13. Lutke-Eversloh T, Steinbuchel A. 2004. Microbial polythioesters. Macromol. Biosci. 4: 166-174.
  14. Yu J-L, Xia X-X, Zhong J-J, Qian Z-G. 2014. Direct biosynthesis of adipic acid from a synthetic pathway in recombinant Escherichia coli. Biotechnol. Bioeng. 111: 2580-2586. https://doi.org/10.1002/bit.25293
  15. Saini M, Wang ZW, Chiang C-J, Chao Y-P. 2014. Metabolic engineering of Escherichia coli for production of butyric acid. J. Agric. Food Chem. 62: 4342-4348. https://doi.org/10.1021/jf500355p
  16. Lutke-Eversloh T, Fischer A, Remminghorst U, Kawada J, Marchessault RH, Bogershausen A, et al. 2002. Biosynthesis of novel thermoplastic polythioesters by engineered Escherichia coli. Nat. Mater. 1: 236-240. https://doi.org/10.1038/nmat773
  17. Xu QS, Shin DH, Pufan R, Yokota H, Kim R, Kim SH. 2004. Crystal structure of a phosphotransacetylase from Streptococcus pyogenes. Proteins 55: 479-481. https://doi.org/10.1002/prot.20039
  18. Xu QS, Jancarik J, Lou Y, Kuznetsova K, Yakunin AF, Yokota H, et al. 2005. Crystal structures of a phosphotransacetylase from Bacillus subtilis and its complex with acetyl phosphate. J. Struct. Funct. Genomics 6: 269-279. https://doi.org/10.1007/s10969-005-9001-9
  19. Iyer PP, Lawrence SH, Luther KB, Rajashankar KR, Yennawar HP, Ferry JG, et al. 2004. Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila. Structure (London, England : 1993) 12: 559-567. https://doi.org/10.1016/j.str.2004.03.007
  20. Lawrence SH, Luther KB, Schindelin H, Ferry JG. 2006. Structural and functional studies suggest a catalytic mechanism for the phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 188: 1143-1154. https://doi.org/10.1128/JB.188.3.1143-1154.2006
  21. Otwinowski Z, Minor W. 1997. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276: 307-326. https://doi.org/10.1016/S0076-6879(97)76066-X
  22. Matthews BW. 1968. Solvent content of protein crystals. J. Mol. Biol. 33: 491-497. https://doi.org/10.1016/0022-2836(68)90205-2
  23. Vagin A, Teplyakov A. 2010. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66: 22-25. https://doi.org/10.1107/S0907444909042589
  24. Emsley P, Cowtan K. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60: 2126-2132. https://doi.org/10.1107/S0907444904019158
  25. Murshudov GN, Skubak 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. https://doi.org/10.1107/S0907444911001314
  26. Laskowski RA, MacArthur MW, Moss DS, Thornton JM. 1993. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26: 283-291. https://doi.org/10.1107/s0021889892009944
  27. Chen VB, Arendall WB, 3rd, 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. https://doi.org/10.1107/S0907444909042073
  28. Lawrence SH, Luther KB, Schindelin H, Ferry JG. 2006. Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from Methanosarcina thermophila. J. Bacteriol. 188: 1143-1154. https://doi.org/10.1128/JB.188.3.1143-1154.2006
  29. Krissinel E, Henrick K. 2007. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372: 774-797. https://doi.org/10.1016/j.jmb.2007.05.022
  30. Wiesenborn DP, Rudolph FB, Papoutsakis ET. 1989. Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis. Appl. Environ. Microbiol. 55: 317-322. https://doi.org/10.1128/aem.55.2.317-322.1989
  31. Zhang Y, Yu M, Yang ST. 2012. Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum. Biotechnol. Progress 28: 52-59. https://doi.org/10.1002/btpr.730
  32. Yoshida Y, Sato M, Nonaka T, Hasegawa Y, Kezuka Y. 2019. Characterization of the phosphotransacetylase-acetate kinase pathway for ATP production in Porphyromonas gingivalis. J. Oral Microbiol. 11: 1588086. https://doi.org/10.1080/20002297.2019.1588086
  33. Thompson JD, Higgins DG, Gibson TJ. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22: 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  34. Ashkenazy H, Abadi S, Martz E, Chay O, Mayrose I, Pupko T, et al. 2016. ConSurf 2016: an improved methodology to estimate and visualize evolutionary conservation in macromolecules. Nucleic Acids Res. 44: W344-350. https://doi.org/10.1093/nar/gkw408