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

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.

Figure 1

Terminal reactions in the pathway of riboflavin biosynthesis. A, 3,4-dihydroxy-2-butanone 4-phosphate synthase; B, 6,7-dimethyl-8-ribityllumazine synthase; C, riboflavin synthase.

Figure 2

Hypothetical reaction mechanism of riboflavin synthase. X, unkown nucleophile; R, ribityl. [11]

Figure 3

Intramolecular sequence similarity of S. pombe riboflavin synthase. N-terminal part, amino acids 1–97; C-terminal part, amino acids 98–208. Identical residues are coloured black. The residues subjected to mutagenesis in this study are marked by an arrow.

Figure 4

Intermediate analogs used for protein perturbation studies. 6,7-bis(trifluoromethyl)-8-ribityllumazine hydrate, epimer A (Compound 6a) and epimer B (Compound 6b). 6-trifluoromethyl-7-oxo-8-ribityllumazine (Compound 7). 6-carboxyethyl-7-oxo-8-ribitylluamzine (Compound 8), 5-nitro-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (Compound 9).

Figure 5

¹⁹F NMR spectra of 6-trifluoromethyl-7-oxo-8-ribityllumazine (Compound 7). A, lumazine protein of P. leiognathi (0.13 mM protein, 0.25 mM 7); B, recombinant N-terminal domain of E. coli riboflavin synthase (0.80 mM protein, 0.98 mM 7); C, riboflavin synthase of E. coli (0.37 mM protein, 1.43 mM 7); D, riboflavin synthase of B. subtilis (0.10 mM protein, 0.75 mM 7); E, riboflavin synthase of S. pombe (0.28 mM protein, 1.22 mM 7). All samples contained 20 mM phosphate buffer, 100 mM KCl and 10% D₂O, pH 7. Signals of the free ligand are indicated by a dashed line. The data were processed with a line broadening of 20 Hz.

Figure 6

¹⁹F NMR signals of 6,7-bis(trifluoromethyl)-8-ribityllumazine hydrate (epimer A; Compound 6a). A, lumazine protein of P. leiognathi (0.14 mM protein, 0.23 mM 6a); B, recombinant N-terminal domain of E. coli riboflavin synthase (0.75 mM protein, 0.56 mM 6a); C, riboflavin synthase of E. coli (0.5 mM protein, 3.34 mM 6a); D, riboflavin synthase of B. subtilis (0.13 mM protein, 0.41 mM 6a); E, riboflavin synthase of S. pombe (0.26 mM protein, 0.60 mM 6a); x, impurities. For other details see legend to Fig. 5.

Figure 7

¹⁹F NMR spectra of 6-trifluoromethyl-7-oxo-8-ribityllumazine (Compound 7) in the presence of riboflavin synthase mutants of S. pombe. Concentrations in the NMR samples were as follows: C48M, 0.28 mM protein, 1.10 mM 7; C48A, 0.15 mM protein, 0.57 mM 7; C48S, 0.15 mM protein, 0.80 mM 7; wild type, 0.28 mM protein, 1.22 mM 7; S146C, 0.20 mM protein, 0.92 mM 7; S146A, 0.26 mM protein, 1 mM 7. For other details see legend to Fig. 5.

Figure 8

¹⁹F NMR spectra of 6,7-bis(trifluoromethyl)-8-ribityllumazine hydrate (epimer A, Compound 6a) in the presence of riboflavin synthase mutants of S. pombe. Concentrations in the NMR samples were as follows: C48M, 0.30 mM protein, 0.94 mM 6a; C48A, 0.19 mM protein, 0.33 mM 6a; C48S, 0.20 mM protein, 0.42 mM 6a; wild type, 0.26 mM protein, 0.60 mM 6a; S146C, 0.26 mM protein, 0.65 mM 6a; S146A, 0.34 mM protein, 1 mM 6a; x, impurities. For other details see legend to Fig. 5.

Figure 9

¹⁹F NMR spectra of 6-trifluoromethyl-7-oxo-8-ribityllumazine (Compound 7) in titration experiments with riboflavin synthase of S. pombe (wild type). The initial concentration of the protein was 0.36 mM. Compound 7 was added to the concentrations indicated. The spectra are displayed with equal intensities of the signals at 15.6 ppm (indicated by *). For other details see legend to Fig. 5.

Figure 10

¹⁹F NMR spectra of 6-trifluoromethyl-7-oxo-8-ribityllumazine (Compound 7) in titration experiments with C48A mutant of riboflavin synthase of S. pombe. The initial concentration of the protein was 0.24 mM. Compound 7 was added to the concentrations indicated in the spectra. The spectra are displayed with equal intensities of the signals as indicated by *. For other details see legend to Fig. 5.