Ligand Binding Properties of the N-Terminal Domain of Riboflavin Synthase from Escherichia coli
Chan Yong Lee1,*, Boris Illarionov2, Young-Eun Woo1,#, Kristina Kemter2, Ryu-Ryun Kim1, Sabine Eberhardt2, Mark Cushman3, Wolfgang Eisenreich2, Markus Fischer4 and Adelbert Bacher2,*
1Department of Biochemistry, Chungnam National University, Daejeon 305-764, Korea
2Lehrstuhl fur Organische Chemie und Biochemie, Technische Universitat Munchen, D-85747 Garching, Germany
3Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, USA
4Lehrstuhl fur Lebensmittelchemie, Universitat Hamburg, D-20146 Hamburg, Germany
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.