Journal of Microbiology and Biotechnology

  • 제30권12호
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  • Pages.1905-1911
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  • 2020
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
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Molecular and Enzymatic Features of Homoserine Dehydrogenase from Bacillus subtilis

  • Kim, Do Hyeon (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Nguyen, Quyet Thang (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Ko, Gyeong Soo (Department of Chemistry, College of Natural Sciences, Soongsil University) ;
  • Yang, Jin Kuk (Department of Chemistry, College of Natural Sciences, Soongsil University)
  • 투고 : 2020.05.01
  • 심사 : 2020.09.25
  • 발행 : 2020.12.28
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초록

Homoserine dehydrogenase (HSD) catalyzes the reversible conversion of ʟ-aspartate-4-semialdehyde to ʟ-homoserine in the aspartate pathway for the biosynthesis of lysine, methionine, threonine, and isoleucine. HSD has attracted great attention for medical and industrial purposes due to its recognized application in the development of pesticides and is being utilized in the large scale production of ʟ-lysine. In this study, HSD from Bacillus subtilis (BsHSD) was overexpressed in Escherichia coli and purified to homogeneity for biochemical characterization. We examined the enzymatic activity of BsHSD for ʟ-homoserine oxidation and found that BsHSD exclusively prefers NADP+ to NAD+ and that its activity was maximal at pH 9.0 and in the presence of 0.4 M NaCl. By kinetic analysis, Km values for ʟ-homoserine and NADP+ were found to be 35.08 ± 2.91 mM and 0.39 ± 0.05 mM, respectively, and the Vmax values were 2.72 ± 0.06 μmol/min-1 mg-1 and 2.79 ± 0.11 μmol/min-1 mg-1, respectively. The apparent molecular mass determined with size-exclusion chromatography indicated that BsHSD forms a tetramer, in contrast to the previously reported dimeric HSDs from other organisms. This novel oligomeric assembly can be attributed to the additional C-terminal ACT domain of BsHSD. Thermal denaturation monitoring by circular dichroism spectroscopy was used to determine its melting temperature, which was 54.8℃. The molecular and biochemical features of BsHSD revealed in this study may lay the foundation for future studies on amino acid metabolism and its application for industrial and medical purposes.

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참고문헌

  1. Jacques SL, Nieman C, Bareich D, Broadhead G, Kinach R, Honek JF, et al. 2001. Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity. Biochim. Biophys. Acta 1544: 28-41. https://doi.org/10.1016/S0167-4838(00)00203-X
  2. Akai S, Ikushiro H, Sawai T, Yano T, Kamiya N, Miyahara I. 2019. The crystal structure of homoserine dehydrogenase complexed with l-homoserine and NADPH in a closed form. J. Biochem. 165: 185-195. https://doi.org/10.1093/jb/mvy094
  3. DeLaBarre B, Thompson PR, Wright GD, Berghuis AM. 2000. Crystal structures of homoserine dehydrogenase suggest a novel catalytic mechanism for oxidoreductases. Nat. Struct. Biol. 7: 238-244. https://doi.org/10.1038/73359
  4. Hayashi J, Inoue S, Kim K, Yoneda K, Kawarabayasi Y, Ohshima T, et al. 2015. Crystal structures of a hyperthermophilic archaeal homoserine dehydrogenase suggest a novel cofactor binding mode for oxidoreductases. Sci. Rep. 5: 11674. https://doi.org/10.1038/srep11674
  5. Navratna V, Reddy G, Gopal B. 2015. Structural basis for the catalytic mechanism of homoserine dehydrogenase. Acta Crystallogr. D Biol. Crystallogr. 71: 1216-1225. https://doi.org/10.1107/S1399004715004617
  6. Tomonaga Y, Kaneko R, Goto M, Ohshima T, Yoshimune K. 2015. Structural insight into activation of homoserine dehydrogenase from the archaeon Sulfolobus tokodaii via reduction. Biochem. Biophys. Rep. 3: 14-17. https://doi.org/10.1016/j.bbrep.2015.07.006
  7. Curien G, Biou V, Mas-Droux C, Robert-Genthon M, Ferrer JL, Dumas R. 2008. Amino acid biosynthesis: new architectures in allosteric enzymes. Plant Physiol. Biochem. 46: 325-339. https://doi.org/10.1016/j.plaphy.2007.12.006
  8. Schuller DJ, Grant GA, Banaszak LJ. 1995. The allosteric ligand site in the Vmax-type cooperative enzyme phosphoglycerate dehydrogenase. Nat. Struct. Biol. 2: 69-76. https://doi.org/10.1038/nsb0195-69
  9. Mas-Droux C, Curien G, Robert-Genthon M, Laurencin M, Ferrer JL, Dumas R. 2006. A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase. Plant Cell 18: 1681-1692. https://doi.org/10.1105/tpc.105.040451
  10. Yoshida A, Tomita T, Kurihara T, Fushinobu S, Kuzuyama T, Nishiyama M. 2007. Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum. J. Mol. Biol. 368: 521-536. https://doi.org/10.1016/j.jmb.2007.02.017
  11. Kaplun A, Vyazmensky M, Zherdev Y, Belenky I, Slutzker A, Mendel S, et al. 2006. Structure of the regulatory subunit of acetohydroxyacid synthase isozyme III from Escherichia coli. J. Mol. Biol. 357: 951-963. https://doi.org/10.1016/j.jmb.2005.12.077
  12. Gallagher DT, Gilliland GL, Xiao G, Zondlo J, Fisher KE, Chinchilla D, et al. 1998. Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase. Structure 6: 465-475. https://doi.org/10.1016/S0969-2126(98)00048-3
  13. Micsonai A, Wien F, Kernya L, Lee YH, Goto Y, Refregiers M, et al. 2015. Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy. Proc. Natl. Acad. Sci. USA 112: E3095-E3103. https://doi.org/10.1073/pnas.1500851112
  14. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, et al. 2000. Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406: 959-964. https://doi.org/10.1038/35023079
  15. Pucci F, Rooman M. 2017. Physical and molecular bases of protein thermal stability and cold adaptation. Curr. Opin. Struct. Biol. 42: 117-128. https://doi.org/10.1016/j.sbi.2016.12.007

피인용 문헌

  1. Expression, purification, and biochemical characterization of an NAD+-dependent homoserine dehydrogenase from the symbiotic Polynucleobacter necessarius subsp. necessarius vol.188, 2020, https://doi.org/10.1016/j.pep.2021.105977