Identification and Characterization of Protein Encoded by orf382 as L-Threonine Dehydrogenase
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
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×His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, L-threonine, ethanol, isopropanol, glucose, glycerol, L-serine, lactic acid, citric acid, methanol, or D-threonine), the enzyme showed the highest activity on L-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of 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 L-threonine dehydrogenase (LTDH). When using L-threonine as the substrate, the enzyme exhibited the best catalytic performance at 39°C and pH 9.8 with NAD+ as the cofactor. The determination of the Km values towards L-threonine (Km = 11.29 μM), ethanol (222.5 μM), and n-butanol (8.02 μM) 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 α-helix percentage (from 12.6% to 6.3%).
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×His-tag protein was then purified and characterized. In an enzymatic assay using different hydroxyl-containing substrates (n-butanol, L-threonine, ethanol, isopropanol, glucose, glycerol, L-serine, lactic acid, citric acid, methanol, or D-threonine), the enzyme showed the highest activity on L-threonine. Characterization of the mutant constructed using gene knockout of the orf382 also implied the function of the enzyme in the metabolism of 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 L-threonine dehydrogenase (LTDH). When using L-threonine as the substrate, the enzyme exhibited the best catalytic performance at 39°C and pH 9.8 with NAD+ as the cofactor. The determination of the Km values towards L-threonine (Km = 11.29 μM), ethanol (222.5 μM), and n-butanol (8.02 μM) 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 α-helix percentage (from 12.6% to 6.3%).
참고문헌 (31)
- Ben-Shitrit T, "Systematic identification of gene annotation errors in the widely used yeast mutation collections", Nat. Methods, 9, pp.373~378 (2012)
- Cochrane G, "The international nucleotide sequence database collaboration", Nucleic Acids Res, 39, pp.15~18 (2011)
- Crow JP, "Sensitivity of the essential zinc-thiolate moiety of yeast alcohol dehydrogenase to hypochlorite and peroxynitrite", Biochemistry, 34, pp.3544~3552 (1995)
- Datsenko KA, "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci", Proc. Natl. Acad. Sci. USA, 97, pp.6640~6645 (2000)
- Feng DD, "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, pp.9915~9923 (2012)
- Greenfield NJ., "Analysis of t he k inetics of f olding o f proteins and peptides using circular dichroism", Nat. Protoc, 1, pp.2891~2899 (2006)
- Greenfield NJ., "Using circular dichroism spectra to estimate protein secondary structure", Nat. Protoc, 1, pp.2876~2890 (2006)
- H ayashi K, "Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110", Mol. Syst. Biol, 2, pp.2006~2007 (2006)
- Hi gashi N, "Kinetic study of thermostable L-threonine dehydrogenase from an archaeon Pyrococcus horikoshii", J. Biosci. Bioeng, 99, pp.175~180 (2005)
- Högbom M, "A high throughput method for the detection of metalloproteins on a microgram scale", Mol. Cell Proteomics, 4, pp.827~834 (2005)
- Jana S, "Effects of guanidine hydrochloride on the conformation and enzyme activity of streptomycin adenylyltransferase monitored by circular dichroism and fluorescence spectroscopy", Biochem. (Mosc.), 71, pp.1230~1237 (2006)
- Johnson AR, "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, pp.382~390 (1996)
- Ka ulmann U, "Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis", Enzyme Microb. Technol, 41, pp.628~637 (2007)
- Kazuoka T, "Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1", J. Bacteriol, 185, pp.4483~4489 (2003)
- Kelly SM, "How to study proteins by circular dichroism", Biochim. Biophys. Acta, 1751, pp.119~139 (2005)
- Kn ight R, "Unlocking the potential of metagenomics through replicated experimental design", Nat. Biotechnol, 30, pp.513~520 (2012)
- Li X, "Sequential extraction of soils for multielement analysis by ICP-AES", Chem. Geol, 124, pp.109~123 (1995)
- Machielsen R, "Production and characterization of a thermostable L-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus", FEBS J, 273, pp.2722~2729 (2006)
- Mardis ER, "A decade’s perspective on DNA sequencing technology", Nature, 470, pp.198~203 (2011)
- Moon TS, "Genetic programs constructed from layered logic gates in single cells", Nature, 491, pp.249~253 (2012)
- N ewman EB, "Role of L-threonine dehydrogenase in the catabolism of threonine and synthesis of glycine by Escherichia coli", J. Bacteriol, 126, pp.1245~1249 (1976)
- Sambrook JR, "Molecular Cloning: A Laboratory Manual, 3rd ed" (2001), Cold Spring Harbor Laboratory Press
- Shimizu Y, "L-Threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 : gene cloning and enzymatic characterization", Extremophiles, 9, pp.317~324 (2005)
- Sibbald MJ, "Mapping the pathways to staphylococcal pathogenesis by comparative secretomics", Microbiol. Mol. Biol. Rev, 70, pp.755~788 (2006)
- Sofia HJ, "Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes", Nucleic Acids Res, 22, pp.2576~2586 (1994)
- Stower H., "Metagenomics: personalized gut microbiome variants", Nat. Rev. Genet, 14, pp.80~ (2013)
- Tressel T, "Interaction between L-threonine dehydrogenase and aminoacetone synthetase and mechanism of aminoacetone production", J. Biol. Chem, 261, pp.16428~16437 (1986)
- Turner JM., "Microbial metabolism of amino ketones. L-1-aminopropan-2-ol dehydrogenase and L-threonine dehydrogenase in Escherichia coli", Biochem. J, 104, pp.112~121 (1967)
- V alnickova Z, "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, pp.1525~1535 (2006)
- Williamson SJ, "From bacterial to microbial ecosystems (metagenomics)", Methods Mol. Biol, 804, pp.35~55 (2012)
- Xu J, "an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp", J. Bacteriol, 177, pp.3166~3175 (1995)