Rationalization of allosteric pathway in Thermus sp. GH5 methylglyoxal synthase
2
조회
0
다운로드
0
인용
0
참고문헌
키워드
초록
A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit,consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS+). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS+, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway. [BMB Reports 2012; 45(12): 748-753]
Abstract
A sequence of 10 amino acids at the C-terminus region of methylglyoxal synthase from Escherichia coli (EMGS) provides an arginine, which plays a crucial role in forming a salt bridge with a proximal aspartate residue in the neighboring subunit,consequently transferring the allosteric signal between subunits. In order to verify the role of arginine, the gene encoding MGS from a thermophile species, Thermus sp. GH5 (TMGS) lacking this arginine was cloned with an additional 30 bp sequence at the 3´-end and then expressed in form of a fusion TMGS with a 10 residual segment at the C-terminus (TMGS+). The resulting recombinant enzyme showed a significant increase in cooperativity towards phosphate, reflected by a change in the Hill coefficient (nH) from 1.5 to 1.99. Experiments including site directed mutagenesis for Asp-10 in TMGS and TMGS+, two dimentional structural survey, fluorescence and irreversible thermoinactivation were carried out to confirm this pathway. [BMB Reports 2012; 45(12): 748-753]
참고문헌 (26)
- Goodey, N. M., "Allosteric regulation and catalysis emerge via a common route", Nat. Chem. Biol., 4, pp.474~482 (2008)
- Summers, M. C., "Proton transfer reactions of methylglyoxal synthase", J. Am. Chem. Soc., 99, pp.4475~4478 (1977)
- Yuan, P. M., "The conversion of dihydroxyacetone phosphate to methylglyoxal and inorganic phosphate by methylglyoxal synthase", Arch. Biochem. Biophys., 183, pp.1~6 (1977)
- Cooper, R. A., "Metabolism of methylglyoxal in microorganisms", Annu. Rev. Microbiol., 38, pp.49~68 (1984)
- Cooper, R. A., "The formation and catabolism of methylglyoxal during glycolysis in Escherichia coli", FEBS Lett., 11, pp.273~276 (1970)
- Marks, G. T., "Mutagenic studies on histidine 98 of methylglyoxal synthase: effects on mechanism and conformational change", Biochemistry, 43, pp.3802~3813 (2004)
- Hopper, D. J., "The purification and properties of Escherichia coli methylglyoxal synthase", Biochem. J., 128, pp.321~329 (1972)
- Saadat, D., "Identification of catalytic bases in the active site of Escherichia coli methylglyoxal synthase: cloning, expression, and functional characterization of conserved aspartic acid residues", Biochemistry, 37, pp.10074~10086 (1998)
- Cooper, R. A., "Methylglyoxal formation during glucose catabolism by Pseudomonas saccharophila. Identification of methylglyoxal synthase", Eur. J. Biochem., 44, pp.81~86 (1974)
- Huang, K., "Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1, 2-propanediol", Appl. Environ. Microbiol., 65, pp.3244~3247 (1999)
- Murata, K., "Characterization of methylglyoxal synthase in Saccharomyces cerevisiae", Biochem. Biophys. Res. Commun., 131, pp.190~198 (1985)
- Ray, S., "Formation of methylglyoxal from aminoacetone by amine oxidase from goat plasma", J. Biol. Chem., 258, pp.3461~3462 (1983)
- Tsai, P. K., "Isolation and characterization of crystalline methylglyoxal synthetase from Proteus vulgaris", J. Biol. Chem., 251, pp.364~367 (1976)
- Cooper, R. A., "Methylglyoxal synthase", Methods. Enzymol., 41, pp.502~508 (1975)
- Marks, G. T., "Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy", Biochemistry, 40, pp.6805~6818 (2001)
- Saadat, D., "The crystal structure of methylglyoxal synthase from Escherichia coli", Structure, 7, pp.309~317 (1999)
- Hopper, D. J., "The regulation of Escherichia coli methylglyoxal synthase; a new control site in glycolysis?", FEBS Lett., 13, pp.213~216 (1971)
- Totemeyer, S., "From famine to feast: the role of methylglyoxal production in Escherichia coli", Mol. Microbiol., 27, pp.553~562 (1998)
- Inoue, Y., "Methylglyoxal and regulation of its metabolism in microorganisms", Adv. Microb. Physiol., 37, pp.177~227 (1995)
- Thornalley, P. J., "The glyoxalase system: new developments towards functional characterization of a metabolic pathway fundamental to biological life", Biochem. J., 269, pp.1~11 (1990)
- Pazhang, M., "Cloning, expression, and characterization of a novel methylglyoxal synthase from Thermus sp. strain GH5", Appl. Biochem. Biotechnol., 162, pp.1519~1528 (2010)
- Sambrook, J., "Molecular cloning: a laboratory manual" (1989), Cold Spring Harbor Laboratory
- Sambrook, J., "Molecular cloning: a laboratory manual" (2001), Cold Spring Harbor Laboratory Press
- Laemmli, U. K., "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", Nature, 227, pp.680~685 (1970)
- Bradford, M. M., "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem., 72, pp.248~254 (1976)
- Wells, C. F., "The spectra of 2,4-Dinitrophenylhydrazone anions and the determination of carbonyl compounds in dilute aqueous solution", Tetrahedron, 22, pp.9~ (1966)