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1.
The biosynthesis of riboflavin precursor 6,7-dimethyl-8-ribityllumazine was studied in extracts of Pichia guilliermondii yeast mutants of rib5 and rib6 genotypes with impaired synthesis of proteins P1 and P2, respectively. It was shown that synthesis of 6,7-dimethyl-8-ribityllumazine took place in extracts of rib5 mutant (active P1 protein) in the presence of 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and the compound formed from ribose-5-phosphate by extracts of rib6 mutant (active P2 protein). No lumazine was formed in extracts of rib6 mutant from pyrimidine substrate and ribose-5-phosphate preincubated with extracts of rib5 mutant. Hence, P1 protein (the product of RIB5 gene) participates in the biosynthesis of 6,7-dimethyl-8-ribityllumazine from 2,4-dihydroxy-5-amino-6-ribitylaminopyrimidine and aliphatic intermediate which is formed from ribose-5-phosphate, under the action of P2 protein (the product of RIB6 gene).  相似文献   

2.
In wild-type cells and some riboflavin-deficient mutants of P. guilliermondii GTP is transformed to the ribitylated intermediates 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 5-amino-2,6-dihydroxy-4-ribitylaminopyrimidine of the riboflavin biosynthetic path. We were able to show that these compounds were formed in vitro as well as in permeabilized cells by reactions including a reductive conversion of the product of GTP cyclohydrolase II action upon GTP. In order to analyse the pyrimidine derivates, 6,7-dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine were synthesized by reaction of pyrimidines with diacetyl. The formation of ribitylated pyrimidines was shown to be strictly dependent on the presence of NADPH2. The data obtained indicate that the reductive step is catalyzed by a 2,5-diamino-6-hydroxy-4-ribosylaminopyrimidine-reductase. 6,7-Dimethyl-8-ribitylpterin and 6,7-dimethyl-8-ribityllumazine isolated from the incubation mixtures have been identified by chromatography and by their ultraviolet and fluorescence spectra.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate. A second transient with an absorption maximum at 445 nm represents an intermediate resulting from the elimination of orthophosphate from the Schiff base. The rate-determining step is the subsequent formation of the 7-exomethylene type anion of 6,7-dimethyl-8-ribityllumazine. The rate constants for the three partial reactions identified by the stopped flow experiments show linear Arrhenius relations in the temperature range of 15-70 degrees C.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
Thirty-nine riboflavin-deficient mutants have been isolated from three yeast strains of Pichia guilliermondii (ATSS 9058, VKM Y-1256, VKM Y-1257) and F5-121 mutant which is capable of production of large amounts of riboflavin in the presence of iron in the medium. All mutants were divided into five groups according to the nature of precursors accumulated in the medium and growth reaction in media with 6,7-dimethyl-8-ribityllumasine and diacetyl. The mutants of the first group did not accumulate specific precursors of riboflavin either in the cells or in the medium. The mutants of the second, third and fourth groups accumulated, after the incubation with diacetyl, 2-amino-4-hydroxy-6,7-dimethylpteridine, 2-amino-4-hydroxy-6,7-dimethyl-8-ribitylpteridine and 6,7-dimethyl-8-ribityllumasine; therefore, they synthesized the following precursors of riboflavin: 2,4,5-triamino-6-hydroxy-pyrimidine, 2,5-diamino-6-hydroxy-4-ribitylaminopyrimidine and 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine. The mutants of the fifth group accumulated 6,7-dimethyl-8-ribityllumasine in the medium and lacked riboflavin synthetase activity, as was confirmed by enzymatic studies.  相似文献   

9.
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.  相似文献   

10.
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.  相似文献   

11.
114 riboflavinless mutants were selected from the genetic line of Pichia guilliermondii yeast. By means of accumulation test the mutants were divided into five biochemical groups. In genetic experiments seven complementation classes were found among 106 mutants. The strains of the I biochemical group, accumulating no specific products, corresponded to complementation class rib1; II group, accumulating 2,4,5-triaminopyrimidine - to the class rib2; III group, accumulating 2,6-dihydroxy-4-ribitylaminopyrimidine - to the class rib3; the mutants of the IV group, accumulating 2,6-dihydroxy-5-amino-4-ribitylaminopyrimidine, were divided into three complementation classes rib4, rib5 and rib6; the mutants of the V group, acculumating 6,7-dimethyl-8-ribityllumazine, corresponded to the class rib7. Two mutants of the IV biochemical group within complementation classes rib4 and rib5 were detected could not grow in the medium with diacetyl without riboflavin. Intragenic complementation was found within classes rib6 and rib7. No linkage between mutations of different complementation classes was detected.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
The conversion of 5-amino-2,4-dioxy-6-ribitylaminopyrimidine (ADRAP) to 6,7-dimethyl-8-ribityllumazine, the immediate precursor of riboflavin, can take place in the presence of an extract of Escherichiacoli. The extract can be separated into 2 protein fraction, both of which are needed for the transformation, and pyridine nucleotide, supplied most efficiently as NAD+, is required. Since no carbon source other than ADRAP is needed, we conclude that 2 moles of ADRAP are used in the transformation, one to serve as donor of the 4 extra carbons needed for the transformation, and one to serve as the acceptor.  相似文献   

16.
6,7-Dimethyl-8-ribityllumazine synthase deficient mutants of Candida guilliermondii were divided into two groups on the basis of in vitro complementation. Mutants of complementation group I produce an intermediate X from ribose 5-phosphate in a reaction requiring Mg++ ions. Compound X was partially purified and was shown to be a phosphoric acid ester. 6,7-Dimethyl-8-ribityllumazine can be formed from Compound X by cell extracts from mutants of complementation group II. The reaction requires 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione or its 5'-phosphate as second substrate. No divalent cations are required.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
Riboflavin is an essential cofactor in all organisms. Its direct biosynthetic precursor, 6,7-dimethyl-8-ribityllumazine, is synthesised by the enzyme 6,7-dimethyl-8-ribityllumazine synthase. Recently, we have found that the enzyme from Schizosaccharomyces pombe binds riboflavin, the final product of the pathway with a relatively high affinity with a KD of 1.2 microM. Here, we report on the crystal structure of lumazine synthase from S. pombe with bound riboflavin and compare the binding mode with those of the substrate analogue inhibitor 5-nitro-6-(D-ribitylamino)-2,4(1H,3H)-pyrimidinedione and of the product analogue 6-carboxyethyl-7-oxo-8-ribityllumazine. In all complexes the pyrimidinedione moieties of each respective ligand bind in a very similar orientation. Binding of riboflavin additionally involves a stacking interaction of the dimethylbenzene moiety with the side-chain of His94, a highly conserved residue in all lumazine synthases. The enzyme from Bacillus subtilis showed a KD of at least 1 mM whereas the very homologous enzyme from Saccharomyces cerevisiae had a comparable KD of 3.9 microM. Structural comparison of the S. cerevisiae, the S. pombe, and the mutant enzymes suggests that fine tuning of affinity is achieved by influencing this stacking interaction.  相似文献   

20.
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.  相似文献   

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