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Spectrofluorometric Characteristics of the N-Terminal Domain of Riboflavin Synthase  

Kim, Ryu-Ryun (Department of Biochemistry, Chungnam National University)
Yi, Jeong-Hwan (Department of Biochemistry, Chungnam National University)
Nam, Ki-Seok (Department of Biochemistry, Chungnam National University)
Ko, Kyung-Won (Department of Biochemistry, Chungnam National University)
Lee, Chan-Yong (Department of Biochemistry, Chungnam National University)
Publication Information
Korean Journal of Microbiology / v.47, no.1, 2011 , pp. 14-21 More about this Journal
Abstract
Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.
Keywords
fluorescence; lumazine; riboflavin; riboflavin synthase; vitamin $B_2$;
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1 Beach, R.L. and G.W. Plaut. 1970. Stereospecificity of the enzymatic synthesis of the o-xylene ring of riboflavin. J. Am. Chem. Soc. 92, 2913-2916.   DOI
2 Eberhardt, S., G. Richter, W. Gimbel, T. Werner, and A. Bacher. 1996. Cloning, sequencing, mapping and hyper-expression of the gene coding for riboflavin synthase of Escherichia coli. Eur. J. Biochem. 242, 712-719.   DOI   ScienceOn
3 Eberhardt, S., N. Zingler, K. Kemter, G. Richter, W. Gimbel, M. Cushman, and A. Bacher. 2001. Domain structure of riboflavin synthase. Eur. J. Biochem. 268, 4315-4323.   DOI   ScienceOn
4 Fischer, M. and A. Bacher. 2005. Biosynthesis of flavocoenzyme. Nat. Prod. Rep. 22, 324-350.   DOI   ScienceOn
5 Fischer, M. and A. Bacher. 2008. Biosynthesis of vitamin B2: Structure and mechanism of riboflavin synthase. Arch. Biochem. Biophys. 474, 252-265.   DOI   ScienceOn
6 Gast, R. and J. Lee. 1978. Isolation of the in vivo emitter in bacterial bioluminescence. Proc. Natl. Acad. Sci. USA 75, 833- 837.   DOI   ScienceOn
7 Illarionov, B., K. Kemter, S. Eberhardt, G. Richter, M. Cushman, and A. Bacher. 2001. Riboflavin synthase of Escherichia coli. Effect of single amino acid substitutions on reaction rate and ligand binding properties. J. Biol. Chem. 276, 11524-11530.   DOI
8 Kim, R.R., B. Illarionov, M. Joshi, M. Cushman, C.Y. Lee, W. Eisenreich, A. Bacher, and M. Fischer. 2010. Mechanistic insights on riboflavin synthase inspired by selective binding of 6,7-dimethyl-8-ribityllumazine exomethylene anion. J. Amer. Chem. Soc. 132, 2983-2980.   DOI   ScienceOn
9 Lee, C.Y., B. Illarionov, Y.E. Woo, K. Kempter, R.R. Kim, S. Eberhardt, M. Cushman, M. Fischer, W. Eisenreich, and A. Bacher. 2007. Ligand binding properties of the riboflavin synthase from Escherichia coli. J. Biochem. Mol. Biol. 40, 239-246.   DOI
10 Liao, D.I., Z. Wawrzak, J.C. Calabrese, P.V. Vitanen, and D.B. Jordan. 2001. Crystal structure of riboflavin synthase. Structure 9, 399-408.   DOI   ScienceOn
11 Marcus, R. and A.M. Coulston. 1990. Water soluble vitamin. The vitamin complex and ascorbic acid. The pharmacological basis of therapeutics, pp. 1534-1536. In Gilman, Rall, Nies, and Tayler (eds.) 8th eds. Pergamon press, New York, USA.
12 Marini, F., A. Naeem, and J.N. Lapeyre. 1993. An efficient 1-tube PCR method for internal site-directed mutagenesis of large amplified molecules. Nucleic Acids Res. 21, 2277-2278.   DOI   ScienceOn
13 Meighen, E.A. 1991. Molecular biology of bacterial bioluminescence. Microbiol. Rev. 55, 123-142.
14 Meining, W., S. Eberhardt, A. Bacher, and R. Ladenstein. 2003. The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6Å resolution. J. Mol. Biol. 331, 1053-1063.   DOI   ScienceOn
15 O'Kane, D.J. and D.C. Prasher. 1992. Evolutionary origins of bacterial bioluminescence. Mol. Microbiol. 6, 443-449.   DOI   ScienceOn
16 O'Kane, D.J., B. Woodward, J. Lee, and D.C. Prasher. 1991. Borrowed proteins in bacterial bioluminescence. Proc. Natl. Acad. Sci. USA 88, 1100-1104.   DOI   ScienceOn
17 Schott, K., J. Kellerman, F. Lottspeich, and A. Bacher. 1990. Riboflavin synthase of Bacillus subtilis. Purification and amino acid sequence of the alpha subunit. J. Biol. Chem. 265, 4204- 4209.
18 Otto, M.K. and A. Bacher. 1981. Ligand-binding studies on light riboflavin synthase from Bacillus subtilis. Eur. J. Biochem. 115, 511-517.
19 Plaut, G.W. 1963. Studies on the nature of the enzymatic conversion of 6,7-dimethyl-8-ribityllumazine to riboflavin. J. Biol. Chem. 238, 2225-2243.
20 Plaut, G.W., R.L. Beach, and T. Aogaichi. 1970. Studies on the mechanism of elimination of protons from the methyl groups of 6,7-dimethyl-8-ribityllumazine by riboflavin synthetase. Biochemistry 9, 771-785.   DOI   ScienceOn
21 Truffault, V., M. Coles, T. Diercks, K. Abelmann, S. Eberhardt, H. Luttgan, A. Bacher, and H. Kessler. 2001. The solution structure of the N-terminal domain of riboflavin synthase. J. Mol. Biol. 309, 949-960.   DOI   ScienceOn
22 Woo, Y.E., S.Y. Kim, and C.Y. Lee. 2005. Expression, and generation of amino-terminal domain of the gene coding for the lumazine protein from Photobacterium phosphoreum. Kor. J. Microbiol. 41, 306-311.