Research article

Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by ¹⁹F NMR protein perturbation experiments

Markus Fischer¹ , Ann-Kathrin Schott¹ , Kristina Kemter¹ , Richard Feicht¹ , Gerald Richter¹ , Boris Illarionov² , Wolfgang Eisenreich¹ , Stefan Gerhardt³ , Mark Cushman⁴ , Stefan Steinbacher³ , Robert Huber³ and Adelbert Bacher¹

¹  Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichten-bergstr. 4, D-85747 Garching, Germany

²  The Institute for Biophysics, Krasnoyarsk, Russia

³  Department of Protein Crystallography, Max-Planck-Institut für Biochemie, Am Klopferspitz 18a, D-82512 Martinsried, Germany

⁴  Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, USA

author email corresponding author email

BMC Biochemistry 2003, 4:18doi:10.1186/1471-2091-4-18

Published:	23 December 2003

Abstract

Background

Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.

Results

A cDNA sequence from Schizosaccharomyces pombe comprising a hypothetical open reading frame with similarity to riboflavin synthase of Escherichia coli was expressed in a recombinant E. coli strain. The recombinant protein is a homotrimer of 23 kDa subunits as shown by sedimentation equilibrium centrifugation. The protein sediments at an apparent velocity of 4.1 S at 20°C. The amino acid sequence is characterized by internal sequence similarity indicating two similar folding domains per subunit. The enzyme catalyzes the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 158 nmol mg^-1 min^-1 with an apparent K_M of 5.7 microM. ¹⁹F NMR protein perturbation experiments using fluorine-substituted intermediate analogs show multiple signals indicating that a given ligand can be bound in at least 4 different states. ¹⁹F NMR signals of enzyme-bound intermediate analogs were assigned to ligands bound by the N-terminal respectively C-terminal folding domain on basis of NMR studies with mutant proteins.

Conclusion

Riboflavin synthase of Schizosaccharomyces pombe is a trimer of identical 23-kDa subunits. The primary structure is characterized by considerable similarity of the C-terminal and N-terminal parts. Riboflavin synthase catalyzes a mechanistically complex dismutation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The ¹⁹F NMR data suggest large scale dynamic mobility in the trimeric protein which may play an important role in the reaction mechanism.