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

BMC Biochem. 2003 Dec 23:4:18. doi: 10.1186/1471-2091-4-18.

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 degrees 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 KM 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Fluorine
  • Molecular Sequence Data
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Pteridines / metabolism
  • Riboflavin / biosynthesis
  • Riboflavin Synthase / chemistry*
  • Riboflavin Synthase / genetics
  • Riboflavin Synthase / metabolism
  • Schizosaccharomyces / enzymology*
  • Sequence Homology, Amino Acid

Substances

  • Pteridines
  • Fluorine
  • 6,7-dimethyl-8-ribityllumazine
  • Riboflavin Synthase
  • Riboflavin