Biosynthesis of riboflavin. Enzymatic formation of 6,7-dimethyl-8-ribityllumazine by heavy riboflavin synthase from Bacillus subtilis

The beta subunits of heavy riboflavin synthase catalyze the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and a carbohydrate phosphate, Compound X. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate is not a substrate for the enzyme, although it is an established intermediate in the biosynthesis of riboflavin. It follows that this pyrimidine phosphate must be dephosphorylated prior to the formation of 6,7-dimethyl-8-ribityllumazine.     

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.mg-1.min-1; Km values for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxybutanone 4-phosphate were 12.5 and 52 micro m, respectively. The enzyme sediments at an apparent velocity of about 12 S. Sedimentation equilibrium analysis indicated a molecular mass around 1 MDa but was hampered by nonideal solute behaviour. Negative-stained electron micrographs showed predominantly spherical particles with a diameter of about 150 A. The data suggest that the enzyme from M. jannaschii can form capsids with icosahedral 532 symmetry consisting of 60 subunits.     

Heavy riboflavin synthase from Bacillus subtilis. Particle dimensions, crystal packing and molecular symmetry

Heavy riboflavin synthase from Bacillus subtilis is an enzyme complex consisting of approximately three alpha-subunits (Mr 23.5 X 10(3)) and 60 beta-subunits (Mr 16 X 10(3)). The enzyme has been crystallized from phosphate buffer in a hexagonal crystal modification that belongs to space group P6(3)22. The asymmetric unit of the crystal cell contains ten beta-subunits. The structure of this unusual 10(6) Mr protein has been studied by small-angle X-ray scattering, electron microscopy of three-dimensional crystals, and crystallographic methods. The scattering curves can be interpreted in terms of a hollow sphere model with a ratio of inner and outer radius of 0.3:1. A diameter of 168 A was estimated from the scattering curves, in close agreement with electron microscopic studies. An aggregate with the stoichiometry beta 60, which was obtained by ligand-driven reaggregation of isolated beta-subunits, showed similar shape and dimensions, but a larger value for the ratio Ri/Ra. Electron micrographs of freeze-etched enzyme crystals showed approximately spherical molecules, which were arranged in hexagonal layers. The lattice constants found from the micrographs are in good agreement with the values derived from X-ray diffraction data. Rotation function calculations in Patterson space showed a set of peaks for 2-fold, 3-fold and 5-fold local rotation axes, accurately consistent with icosahedral symmetry and with the particle orientation A shown in the Appendix. The crystal packing can be described as follows: enzyme particles with icosahedral symmetry (point group 532) are located at points 32 of the hexagonal cell, corresponding to positions (0, 0, 0) and (0, 0, 1/2) on the 6-fold screw axes. From the data reported, it may be concluded that the enzyme structure can be described as an icosahedral capsid of 60 beta-subunits with the triangulation number T = 1. The alpha-subunits are located in the central core space of the capsid, but their spatial orientation is incompletely understood.     

The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6A resolution

Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity. A recombinant peptide comprising amino acid residues 1-97 forms a dimer, which binds riboflavin with high affinity. Here, we report the structure of this construct in complex with riboflavin at 2.6A resolution. It is demonstrated that the complex can serve as a model for ligand-binding in the native enzyme. The structure and riboflavin-binding mode is in excellent agreement with structural information obtained from the native enzyme from Escherichia coli and riboflavin synthase from Schizosaccharomyces pombe. The implications for the binding specificity and the regiospecificity of the catalyzed reaction are discussed.     

Studies on the reaction mechanism of riboflavin synthase: X-ray crystal structure of a complex with 6-carboxyethyl-7-oxo-8-ribityllumazine

Riboflavin synthase catalyzes the disproportionation of 6,7-dimethyl-8-ribityllumazine affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. We have determined the structure of riboflavin synthase from Schizosaccharomyces pombe in complex with the substrate analog, 6-carboxyethyl-7-oxo-8-ribityllumazine at 2.1 A resolution. In contrast to the homotrimeric solution state of native riboflavin synthase, we found the enzyme to be monomeric in the crystal structure. Structural comparison of the riboflavin synthases of S. pombe and Escherichia coli suggests oligomer contact sites and delineates the catalytic site for dimerization of the substrate and subsequent fragmentation of the pentacyclic intermediate. The pentacyclic substrate dimer was modeled into the proposed active site, and its stereochemical features were determined. The model suggests that the substrate molecule at the C-terminal domain donates a four-carbon unit to the substrate molecule bound at the N-terminal domain of an adjacent subunit in the oligomer.     

Biosynthesis of riboflavin: 6,7-dimethyl-8-ribityllumazine synthase of Schizosaccharomyces pombe.

A cDNA sequence from Schizosaccharomyces pombe with similarity to 6,7-dimethyl-8-ribityllumazine synthase was expressed in a recombinant Escherichia coli strain. The recombinant protein is a homopentamer of 17-kDa subunits with an apparent molecular mass of 87 kDa as determined by sedimentation equilibrium centrifugation (it sediments at an apparent velocity of 5.0 S at 20 degrees C). The protein has been crystallized in space group C2221. The crystals diffract to a resolution of 2.4 A. The enzyme catalyses the formation of 6,7-dimethyl-8-ribityllumazine from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy- 2-butanone 4-phosphate. Steady-state kinetic analysis afforded a vmax value of 13 000 nmol.mg-1.h-1 and Km values of 5 and 67 microm for 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, respectively. The enzyme binds riboflavin with a Kd of 1.2 microm. The fluorescence quantum yield of enzyme-bound riboflavin is < 2% as compared with that of free riboflavin. The protein/riboflavin complex displays an optical transition centered around 530 nm as shown by absorbance and CD spectrometry which may indicate a charge transfer complex. Replacement of tryptophan 27 by tyrosine or phenylalanine had only minor effects on the kinetic properties, but complexes of the mutant proteins did not show the anomalous long wavelength absorbance of the wild-type protein. The replacement of tryptophan 27 by aliphatic amino acids substantially reduced the affinity of the enzyme for riboflavin and for the substrate, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.     

The lumazine synthase-riboflavin synthase complex of Bacillus subtilis. Crystallization of reconstituted icosahedral beta-subunit capsids

The lumazine synthase-riboflavin synthase complex (heavy riboflavin synthase) of Bacillus subtilis consists of an icosahedral capsid of 60 beta-subunits containing a core of three alpha-subunits. The enzyme has been purified from the derepressed mutant H 94 of B. subtilis by a novel efficient procedure using column chromatography and preparative crystallization. Beta-Subunits were isolated after dissociation of the enzyme at pH 8.0. Ligand-driven renaturation of beta-subunits yields hollow icosahedral beta 60 capsids which could be crystallized in 1.55 M phosphate, pH 8.7, in three different modifications. A monoclinic modification belongs to space group C2 with unit cell dimensions of a = 235.5, b = 191.2, and c = 165.4 A and alpha = gamma = 90 degrees and beta = 134.4 degrees. The crystals contain two hollow beta 60 particles/unit cell and diffract to approximately 2.8-A resolution. A hexagonal modification has the space group P6(3)22 with unit cell dimensions of a = b = 157.2 and c = 300.8 A and alpha = beta = 90 degrees and gamma = 120 degrees. These cell parameters are similar to the dimensions of hexagonal crystals of native heavy riboflavin synthase (alpha 3 beta 60). A second hexagonal modification shows unit cell parameters of a = b = 156.3 and c = 622.6 A and alpha = beta = 90 degrees and gamma = 120 degrees. The space group of this modification could not be determined unambiguously.     

Pre-steady-state kinetic analysis of riboflavin synthase using a pentacyclic reaction intermediate as substrate

Riboflavin synthase catalyses a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H )-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. A pentacyclic adduct (compound 2 ) of two substrate molecules was used as substrate for pre-steady-state kinetic analysis. Whereas the wild-type enzyme catalyses the decomposition of compound 2 into a mixture of riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H )-pyrimidinedione, as well as into two equivalents of 6,7-dimethyl-8-ribityllumazine, a H102Q mutant enzyme predominantly catalyses the former reaction. Stopped-flow experiments with this mutant enzyme failed to identify a reaction intermediate between compound 2 and riboflavin. However, the apparent rate constants for the formation of riboflavin as observed by stopped-flow and quenched-flow experiments were significantly different, thus suggesting that the reaction proceeds via a significantly populated intermediate, the absorbance of which is similar to that of compound 2 . An F2A mutant enzyme converts compound 2 predominantly into 6,7-dimethyl-8-ribityllumazine. Stopped-flow experiments using compound 2 as substrate indicated a slight and rapid initial increase in absorbance at 310 nm, followed by a slower decrease. This finding, in conjunction with different apparent rates for the formation of 6,7-dimethyl-8-ribityllumazine, suggests the involvement of a significantly populated intermediate in the transition between compound 2 and 6,7-dimethyl-8-ribityllumazine, the optical spectrum of which is similar to that of compound 1.     

Biosynthesis of vitamin B2: diastereomeric reaction intermediates of archaeal and non-archaeal riboflavin synthases

The dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase affords riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. A pentacyclic adduct of two 6,7-dimethyl-8-ribityllumazines has been identified earlier as a catalytically competent reaction intermediate of the Escherichia coli enzyme. Acid quenching of reaction mixtures of riboflavin synthase of Methanococcus jannaschii, a paralog of 6,7-dimethyl-8-ribityllumazine synthase devoid of similarity with riboflavin synthases of eubacteria and eukaryotes, afforded a compound whose optical absorption and NMR spectra resemble that of the pentacyclic E. coli riboflavin synthase intermediate, whereas the circular dichroism spectra of the two compounds have similar envelopes but opposite signs. Each of the compounds could serve as a catalytically competent intermediate for the enzyme by which it was produced, but not vice versa. All available data indicate that the respective pentacyclic intermediates of the M. jannaschii and E. coli enzymes are diastereomers.     

Presteady state kinetic analysis of riboflavin synthase

Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate. No other transient species were sufficiently populated to allow detection. The data are best described by a sequence of one second order and one first order reaction.     

Biosynthesis of vitamin B2 in plants

The biosynthesis of one riboflavin (vitamin B₂) molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 2,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate yields 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione, which is recycled in the biosynthetic pathway. Characteristic architectural features of most enzymes involved in the plant riboflavin pathway resemble those of eubacteria, whereas the similarities between plants and yeasts are less pronounced. Moreover, riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a somewhat different pathway.     

Structural basis of charge transfer complex formation by riboflavin bound to 6,7-dimethyl-8-ribityllumazine synthase.

The amino acid residue tryptophan 27 of 6,7-dimethyl-8-ribityllumazine synthase of the yeast Schizosaccharomyces pombe was replaced by tyrosine. The structures of the W27Y mutant protein in complex with riboflavin, the substrate analogue 5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, and the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine, were determined by X-ray crystallography at resolutions of 2.7-2.8 A. Whereas the indole system of W27 forms a coplanar pi-complex with riboflavin, the corresponding phenyl ring in the W27Y mutant establishes only peripheral contact with the heterocyclic ring system of the bound riboflavin. These findings provide an explanation for the absence of the long wavelength shift in optical absorption spectra of riboflavin bound to the mutant enzyme. The structures of the mutants are important tools for the interpretation of the unusual physical properties of riboflavin in complex with lumazine synthase.     

Studies on the 4-carbon precursor in the biosynthesis of riboflavin. Purification and properties of L-3,4-dihydroxy-2-butanone-4-phosphate synthase

The formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione requires a phosphorylated 4-carbon intermediate which has been designated as Compound X (Neuberger, G., and Bacher, A. (1985) Biochem. Biophys. Res. Commun. 127, 175-181). The enzyme catalyzing the formation of Compound X has been purified about 600-fold from the cell extract of the flavinogenic yeast Candida guilliermondii by chromatographic procedures. The purified protein appeared homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of a single polypeptide of 24 kDa. The committed substrate of the enzyme was identified as D-ribulose 5-phosphate. The enzyme yields two products which were identified as L-3,4-dihydroxy-2-butanone 4-phosphate and formate by NMR and CD spectroscopy. Mg2+ is required for activity.     

The pyrimidine nucleotide reductase step in riboflavin and F(420) biosynthesis in archaea proceeds by the eukaryotic route to riboflavin

The Methanococcus jannaschii gene MJ0671 was cloned and overexpressed in Escherichia coli, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (compound 5). Only compound 3 was found to serve as a substrate for the enzyme. NADPH and NADH functioned equally well as the reductants. This specificity for the reduction of compound 3 was also confirmed by using cell extracts of M. jannaschii and Methanosarcina thermophila. Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts. The absence of the other genes in the biosynthesis of riboflavin in Archaea is discussed.     

Biosynthesis of vitamin B2: Structure and mechanism of riboflavin synthase

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. GTP is hydrolytically opened, converted into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate leads to 6,7-dimethyl-8-ribityllumazine. The final step in the biosynthesis of the vitamin involves the dismutation of 6,7-dimethyl-8-ribityllumazine catalyzed by riboflavin synthase. The mechanistically unusual reaction involves the transfer of a four-carbon fragment between two identical substrate molecules. The second product, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, is recycled in the biosynthetic pathway by 6,7-dimethyl-8-ribityllumazine synthase. This article will review structures and reaction mechanisms of riboflavin synthases and related proteins up to 2007 and 122 references are cited.     

Biosynthetic precursors of deazaflavins.

The incorporation of 13C- and 14C-labeled precursors into 5-deaza-7,8-didemethyl-8-hydroxyriboflavin (factor F0) was studied with growing cells of Methanobacterium thermoautotrophicum. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was incorporated into the deazaflavin and into riboflavin without dilution. Tyrosine and 4-hydroxyphenylpyruvate were incorporated into the deazaflavin and into cellular protein. 4-Hydroxybenzaldehyde was not incorporated. A reaction mechanism is proposed for the formation of the deazaflavin chromophore from 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and tyrosine or 4-hydroxyphenylpyruvate.     

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.     

Enzyme catalysis via control of activation entropy: site-directed mutagenesis of 6,7-dimethyl-8-ribityllumazine synthase

6,7-Dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyses the penultimate step in the biosynthesis of riboflavin. In Bacillus subtilis, 60 lumazine synthase subunits form an icosahedral capsid enclosing a homotrimeric riboflavin synthase unit. The ribH gene specifying the lumazine synthase subunit can be expressed in high yield. All amino acid residues exposed at the surface of the active site cavity were modified by PCR assisted mutagenesis. Polar amino acid residues in direct contact with the enzyme substrates, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate, could be replaced with relative impunity with regard to the catalytic properties. Only the replacement of Arg127, which forms a salt bridge with the phosphate group of 3,4-dihydroxy-2-butanone 4-phosphate, reduced the catalytic rate by more than one order of magnitude. Replacement of His88, which is believed to assist in proton transfer reactions, reduced the catalytic activity by about one order of magnitude. Surprisingly, the activation enthalpy deltaH of the lumazine synthase reaction exceeds that of the uncatalysed reaction. On the other hand, the free energy of activation deltaG of the uncatalysed reaction is characterised by a large entropic term (TdeltaS) of -37.8 kJmol(-1), whereas the entropy of activation (TdeltaS) of the enzyme-catalysed reaction is -6.7 kJmol(-1). This suggests that the rate enhancement by the enzyme is predominantly achieved by establishing a favourable topological relation of the two substrates, whereas acid/base catalysis may play a secondary role.     

Evolution of vitamin B2 biosynthesis: 6,7-dimethyl-8-ribityllumazine synthases of Brucella

The penultimate step in the biosynthesis of riboflavin (vitamin B2) involves the condensation of 3,4-dihydroxy-2-butanone 4-phosphate with 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase). Pathogenic Brucella species adapted to an intracellular lifestyle have two genes involved in riboflavin synthesis, ribH1 and ribH2, which are located on different chromosomes. The ribH2 gene was shown previously to specify a lumazine synthase (type II lumazine synthase) with an unusual decameric structure and a very high Km for 3,4-dihydroxy-2-butanone 4-phosphate. Moreover, the protein was found to be an immunodominant Brucella antigen and was able to generate strong humoral as well as cellular immunity against Brucella abortus in mice. We have now cloned and expressed the ribH1 gene, which is located inside a small riboflavin operon, together with two other putative riboflavin biosynthesis genes and the nusB gene, specifying an antitermination factor. The RibH1 protein (type I lumazine synthase) is a homopentamer catalyzing the formation of 6,7-dimethyl-8-ribityllumazine at a rate of 18 nmol mg(-1) min(-1). Sequence comparison of lumazine synthases from archaea, bacteria, plants, and fungi suggests a family of proteins comprising archaeal lumazine and riboflavin synthases, type I lumazine synthases, and the eubacterial type II lumazine synthases.     

Biosynthesis of riboflavin. Enzymatic formation of the xylene moiety from [14C]ribulose 5-phosphate

We have studied the enzymatic formation of the xylene ring of riboflavin using cell extracts from the flavinogenic yeast Candida guilliermondii. 5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5′-phosphate could serve as substrates. In addition, a pentose phosphate or pentulose phosphate was required. Experiments with [¹⁴C]ribulose 5-phosphate gave evidence for the incorporation of the ribulose carbon atoms except C-4 into the xylene ring of the vitamin.