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KMID : 0380220070400020239
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Journal of Biochemistry and Molecular Biology
2007 Volume.40 No. 2 p.239 ~ p.246
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Ligand Binding Properties of the N-Terminal Domain of Riboflavin Synthase from Escherichia coli
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Lee Chan-Yong
Illarionov Boris
Kemter Kristina
Woo Young-Eun
Kim Ryu-Ryun
Eberhardt Sabine
Cushman Mark
Eisenreich Wolfgang
Fischer Markus
Bacher Adelbert
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Abstract
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Riboflavin synthase from Escherichia coli is a homotrimer of 23.4 kDa subunits and catalyzes the formation of one molecule each of riboflavin and 5-amino-6-ribitylamino- 2,4(1H,3H)-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrate, 6,7- dimethyl-8-ribityllumazine. Each subunit comprises two closely similar folding domains. Recombinant expression of the N-terminal domain is known to provide a c2-symmetric homodimer. In this study, the binding properties of wild type as well as two mutated proteins of N-terminal domain of riboflavin synthase with various ligands were tested. The replacement of the amino acid residue A43, located in the second shell of riboflavin synthase active center, in the recombinant N-terminal domain dimer reduces the affinity for 6,7-dimethyl-8-ribityllumazine. The mutation of the amino acid residue C48 forming part of activity cavity of the enzyme causes significant 19F NMR chemical shift modulation of trifluoromethyl derivatives of 6,7-dimethyl-8-ribityllumazine in complex with the protein, while substitution of A43 results in smaller chemical shift changes.
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KEYWORD
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Escherichia coli, Lumazine, Riboflavin synthase, Site-directed mutagenesis
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