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


CONCLUSIONS
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.
Fischer
Markus
M
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichten-bergstr, 4, D-85747 Garching, Germany. markus.fischer@ch.tum.de
Schott
Ann-Kathrin
AK
Kemter
Kristina
K
Feicht
Richard
R
Richter
Gerald
G
Illarionov
Boris
B
Eisenreich
Wolfgang
W
Gerhardt
Stefan
S
Cushman
Mark
M
Steinbacher
Stefan
S
Huber
Robert
R
Bacher
Adelbert
A
D000595:Amino Acid Sequence; D005461:Fluorine; D008969:Molecular Sequence Data; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011621:Pteridines / Q000378:metabolism; D012256:Riboflavin / Q000096:biosynthesis; D012258:Riboflavin Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid
D011621:Pteridines; D005461:Fluorine; C001061:6,7-dimethyl-8-ribityllumazine; D012258:Riboflavin Synthase; D012256:Riboflavin