Journal of Microbiology and Biotechnology

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

DOI QR Code

Identification and Characterization of Protein Encoded by orf382 as $\small{L}$-Threonine Dehydrogenase

  • Ma, Fei (State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology) ;
  • Wang, Tianwen (State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology) ;
  • Ma, Xingyuan (State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology) ;
  • Wang, Ping (State Key Laboratory of Bioreactor Engineering, Biomedical Nanotechnology Center, School of Biotechnology, East China University of Science and Technology)
  • Received : 2013.12.11
  • Accepted : 2014.03.18
  • Published : 2014.06.28
Download PDF
⟨ Previous Next ⟩

Abstract

In the genome annotation of Escherichia coli MG1655, the orf382 (1,149 bp) is designated as a gene encoding an alcohol dehydrogenase that may be Fe-dependent. In this study, the gene was amplified from the genome by PCR and overexpressed in Escherichia coli BL21(DE3). The recombinant $6{\times}$His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, $\small{L}$-threonine, ethanol, isopropanol, glucose, glycerol, $\small{L}$-serine, lactic acid, citric acid, methanol, or $\small{D}$-threonine), the enzyme showed the highest activity on $\small{L}$-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of $\small{L}$-threonine into glycine. Considering the presence of tested substrates in living E. coli cel ls and previous literature, we believed that the suitable nomenclature for the enzyme should be an $\small{L}$-threonine dehydrogenase (LTDH). When using $\small{L}$-threonine as the substrate, the enzyme exhibited the best catalytic performance at $39^{\circ}C$ and pH 9.8 with $NAD^+$ as the cofactor. The determination of the Km values towards $\small{L}$-threonine (Km = $11.29{\mu}M$), ethanol ($222.5{\mu}M$), and n-butanol ($8.02{\mu}M$) also confirmed the enzyme as an LTDH. Furthermore, the LTDH was shown to be an ion-containing protein based on inductively coupled plasma-atomic emission spectrometry with an isoelectronic point of pH 5.4. Moreover, a circular dichroism analysis revealed that the metal ion was structurally and enzymatically essential, as its deprivation remarkably changed the ${\alpha}$-helix percentage (from 12.6% to 6.3%).

Keywords

References

  1. B en-Shitrit T, Yosef N, Shemesh K, Sharan R, Ruppin E, Kupiec M. 2012. Systematic identification of gene annotation errors in the widely used yeast mutation collections. Nat. Methods 9: 373-378. https://doi.org/10.1038/nmeth.1890
  2. Cochrane G, Karsch-Mizrachi I, Nakamura Y. 2011. The international nucleotide sequence database collaboration. Nucleic Acids Res. 39: D15-D18. https://doi.org/10.1093/nar/gkq1150
  3. Crow JP, Beckman JS, McCord JM. 1995. Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite. Biochemistry 34: 3544-3552. https://doi.org/10.1021/bi00011a008
  4. Datsenko KA, Wanner BL. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. USA 97: 6640-6645. https://doi.org/10.1073/pnas.120163297
  5. Feng DD, Yang W, Yang WJ, Yu QY, Zhang Z. 2012. Molecular cloning, heterogenous expression and the induction profiles after organophosphate phoxim exposure of the carboxylesterase Bmae33 in the silkworm, Bombyx mori. Afr. J. Biotechnol. 11: 9915-9923.
  6. Greenfield NJ. 2006. Analysis of t he k inetics of f olding o f proteins and peptides using circular dichroism. Nat. Protoc. 1: 2891-2899.
  7. Greenfield NJ. 2006. Using circular dichroism spectra to estimate protein secondary structure. Nat. Protoc. 1: 2876- 2890.
  8. H ayashi K, Morooka N, Yamamoto Y, Fujita K, Isono K, Choi S, et al. 2006. Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2: 2006-2007.
  9. Hi gashi N, Fukada H, Ishikawa K. 2005. Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii. J. Biosci. Bioeng. 99: 175-180. https://doi.org/10.1263/jbb.99.175
  10. Ho gbom M, Lam R, Bakali HMA, Kuznetsova E, Nordlund P, Z amble DB. 2005. A h igh throughput method for the detection of metalloproteins on a microgram scale. Mol. Cell Proteomics 4: 827-834. https://doi.org/10.1074/mcp.T400023-MCP200
  11. Jana S, Chaudhuri TK, Deb JK. 2006. Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy. Biochem. (Mosc.) 71: 1230-1237. https://doi.org/10.1134/S0006297906110083
  12. Johnson AR, Dekker EE. 1996. Woodward's reagent K inactivation of Escherichia coli L-threonine dehydrogenase: increased absorbance at 340-350 nm is due to modification of cysteine and histidine residues, not aspartate or glutamate carboxyl groups. Protein Sci. 5: 382-390.
  13. Ka ulmann U, Smithies K, Smith MEB, Hailes HC, Ward JM. 2007. Substrate spectrum of ${\omega}g$-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb. Technol. 41: 628-637. https://doi.org/10.1016/j.enzmictec.2007.05.011
  14. K azuoka T, Takigawa S, Arakawa N, Hizukuri Y, Muraoka I, O ikawa T, Soda K. 2003. Novel p sychrophilic a nd thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1. J. Bacteriol. 185: 4483-4489. https://doi.org/10.1128/JB.185.15.4483-4489.2003
  15. Kelly SM, Jess TJ, Price NC. 2005. How to study proteins by circular dichroism. Biochim. Biophys. Acta 1751: 119-139. https://doi.org/10.1016/j.bbapap.2005.06.005
  16. Kn ight R, Jansson J, Field D, Fierer N, Desai N, Fuhrman JA, et al. 2012. Unlocking the potential of metagenomics through replicated experimental design. Nat. Biotechnol. 30: 513-520. https://doi.org/10.1038/nbt.2235
  17. Li X, Coles BJ, Ramsey MH, Thornton I. 1995. Sequential extraction of soils for multielement analysis by ICP-AES. Chem. Geol. 124: 109-123. https://doi.org/10.1016/0009-2541(95)00029-L
  18. Machielsen R, van der Oost J. 2006. Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. FEBS J. 273: 2722-2729. https://doi.org/10.1111/j.1742-4658.2006.05290.x
  19. Mardis ER. 2011. A decade's perspective on DNA sequencing technology. Nature 470: 198-203. https://doi.org/10.1038/nature09796
  20. M oon TS, Lou C, Tamsir A, Stanton BC, Voigt CA. 2012. Genetic programs constructed from layered logic gates in single cells. Nature 491: 249-253. https://doi.org/10.1038/nature11516
  21. N ewman EB, Kapoor V, Potter R. 1976. Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli. J. Bacteriol. 126: 1245-1249.
  22. Sambrook J R, R ussell D W. 2001. Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York.
  23. Shimizu Y, Sakuraba H, Kawakami R, Goda S, Kawarabayasi Y, O hshima T . 2005. L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3: gene cloning and enzymatic characterization. Extremophiles 9: 317-324. https://doi.org/10.1007/s00792-005-0447-2
  24. Si bbald MJ, Ziebandt AK, Engelmann S, Hecker M, de Jong A, Harmsen HJ, et al. 2006. Mapping t he p athways to staphylococcal pathogenesis by comparative secretomics. Microbiol. Mol. Biol. Rev. 70: 755-788. https://doi.org/10.1128/MMBR.00008-06
  25. Sofi a HJ, Burland V, Daniels DL, Plunkett G 3rd, Blattner FR. 1994. Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes. Nucleic Acids Res. 22: 2576-2586. https://doi.org/10.1093/nar/22.13.2576
  26. St ower H. 2013. Metagenomics: personalized gut microbiome variants. Nat. Rev. Genet. 14: 80. https://doi.org/10.1038/nrg3409
  27. T ressel T, Thompson R, Zieske LR, Menendez MI, Davis L. 1986. Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production. J. Biol. Chem. 261: 16428-16437.
  28. Turner JM. 1967. Microbial metabolism of amino ketones. L- 1-aminopropan-2-ol dehydrogenase and L-threonine dehydrogenase in Escherichia coli. Biochem. J. 104: 112-121. https://doi.org/10.1042/bj1040112
  29. V alnickova Z, Christensen T, Skottrup P, Thogersen IB, Hojrup P, Enghild JJ. 2006. Post-translational modifications of human thrombin-activatable fibrinolysis inhibitor (TAFI): evidence for a large shift in the isoelectric point and reduced solubility upon activation. Biochemistry 45: 1525-1535. https://doi.org/10.1021/bi051956v
  30. Williamson SJ, Yooseph S. 2012. From bacterial to microbial ecosystems (metagenomics). Methods Mol. Biol. 804: 35-55. https://doi.org/10.1007/978-1-61779-361-5_3
  31. Xu J , Johnson R C. 1995. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J. Bacteriol. 177: 3166- 3175. https://doi.org/10.1128/jb.177.11.3166-3175.1995

Cited by

  1. Use of an Alternative Pathway for Isoleucine Synthesis in Threonine-Producing Strains of Escherichia coli vol.56, pp.7, 2014, https://doi.org/10.1134/s0003683820070066