Ligand binding properties of the N-terminal domain of riboflavin synthase from Escherichia coli.
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 c(2)-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 (19)F 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.
Lee
Chan Yong
CY
Lehrstuhl fur Organische Chemie und Biochemie, Technische Universitat Munchen, D-85747 Garching, Germany. adelbert.bacher@ch.tum.de
Illarionov
Boris
B
Woo
Young-Eun
YE
Kemter
Kristina
K
Kim
Ryu-Ryun
RR
Eberhardt
Sabine
S
Cushman
Mark
M
Eisenreich
Wolfgang
W
Fischer
Markus
M
Bacher
Adelbert
A
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012258:Riboflavin Synthase / Q000737:chemistry* / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D017186:Titrimetry
D008024:Ligands; D050505:Mutant Proteins; D012258:Riboflavin Synthase