Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes.

In this paper, we report the identification, cloning, and complete nucleotide sequence of four genes from Actinobacillus pleuropneumoniae that are involved in riboflavin biosynthesis. The cloned genes can specify production of large amounts of riboflavin in Escherichia coli, can complement several defined genetic mutations in riboflavin biosynthesis in E. coli, and are homologous to riboflavin biosynthetic genes from E. coli, Haemophilus influenzae, and Bacillus subtilis. The genes have been designated A. pleuropneumoniae ribGBAH because of their similarity in both sequence and arrangement to the B. subtilis ribGBAH operon.     

On the biosynthesis of 5-methoxybenzimidazole. Precursor-function of 5-hydroxybenzimidazole, benzimidazole and riboflavin.

1. In Clostridium thermoaceticum 5-hydroxybenzimidazole is methylated to 5-methoxybenz-imidaxole and transformed to 5-methoxybenzimidazolylcobamide. 5-Hydroxybenzimidazolycobamide is also methylated to 5-methoxybenzimidazolylcobamide. These results indicate a possible precursor function of 5-hydroxybenzimidazole in the biosynthesis of 5-methoxybenzimidazole. 2. The same microorganism uses benzimidazole to form benzimidazolylcobamide. This or externally added benzimidazolylcobamide, although taken up by the cells, is not further transformed (i.e. hydroxylated and methylated to 5-methoxybenzimdazolylcobamidel). This excludes a precursor function of benzimidazole in the biosynthesis of 5-methoxybenzimidazole. 3. Contrary to the biosynthesis of 5,6-dimethylbenzimidazole, 5-methoxybenzimidazole is not formed from riboflavin, but riboflavin inhibits the growth and the production of 5-methoxybenzimidazolylcobamide in Clostridium thermoaceticum. A tentative scheme for the biosynthesis of 5-methoxybenzimidazole via a riboflavin analog is discussed.     

Operon of riboflavin biosynthesis in Bacillus subtilis. XVII. A study of the regulatory functions of the intermediate products and their derivatives

Repression of synthesis of GTP-cyclohydrolase and riboflavinsynthetase was studied in different regulatory mutants of Bacillus subtilis. The results of experiments with some riboflavin precursors and their derivatives revealed that 5-amino-2,6-dioxo-4-ribitylaminopyrimidine and 6-methyl-7-(1',2'-dioxyethyl)-8-ribityllumazine can serve as effectors in riboflavin biosynthesis.     

Operon of riboflavin biosynthesis in Bacillus subtilis. XVI. Localization of the ribC-group markers on the chromosome]

Regulatory markers of ribC group were located on the chromosome of Bacillus subtilis by means of genetic transformation. Markers of this group controlling the regulation of riboflavin biosynthesis were mapped between markers of resistance to acriflavin and streptomycin (strC group). The value of cotransfer index between acriflavin-resistance markers and ribC markers was found to be 26--32%. Acriflavin inhibits the riboflavin biosynthesis. The level of inhibition depends on the genotype of riboflavin-producing strains, while the inhibition of the cell growth does not depend on it.     

Riboflavin analogs and inhibitors of riboflavin biosynthesis

Flavins are active components of many enzymes. In most cases, riboflavin (vitamin B₂) as a coenzyme represents the catalytic part of the holoenzyme. Riboflavin is an amphiphatic molecule and allows a large variety of different interactions with the enzyme itself and also with the substrate. A great number of active riboflavin analogs can readily be synthesized by chemical methods and, thus, a large number of possible inhibitors for many different enzyme targets is conceivable. As mammalian and especially human biochemistry depends on flavins as well, the target of the inhibiting flavin analog has to be carefully selected to avoid unwanted effects. In addition to flavoproteins, enzymes, which are involved in the biosynthesis of flavins, are possible targets for anti-infectives. Only a few flavin analogs or inhibitors of flavin biosynthesis have been subjected to detailed studies to evaluate their biological activity. Nevertheless, flavin analogs certainly have the potential to serve as basic structures for the development of novel anti-infectives and it is possible that, in the future, the urgent need for new molecules to fight multiresistant microorganisms will be met.     

Cloning of the Bacillus subtilis DNA fragment containing the genes for lysine and riboflavin biosynthesis

The SalI fragment of chromosomal DNA of Bacillus subtilis carrying the gene for lysine biosynthesis and the regulatory operator region (ribO) from the riboflavin gene was cloned into Escherichia coli cells. This fragment was shown to contain the gene coding for lysine synthesizing enzyme. Localization of this gene in Bac. subtili was determined. New plasmids pLRS33 and pLRB4 were constructed using pBR322; they carry a fragment homologous to pLP102 plasmid containing the operon for riboflavin biosynthesis.     

Production of riboflavin by metabolically engineered Corynebacterium ammoniagenes

Improved strains for the production of riboflavin (vitamin B₂) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5′-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l⁻¹ for 72 h in a 5-l jar fermentor without any end product inhibition. Received: 23 August 1999 / Received revision: 13 October 1999 / Accepted: 5 November 1999     

Evolution of vitamin B2 biosynthesis. A novel class of riboflavin synthase in Archaea

The open reading frame MJ1184 of Methanococcus jannaschii with similarity to riboflavin synthase of Methanothermobacter thermoautotrophicus was cloned into an expression vector but was poorly expressed in an Escherichia coli host strain. However, a synthetic open reading frame that was optimized for expression in E.coli directed the synthesis of abundant amounts of a protein with an apparent subunit mass of 17.5 kDa. The protein was purified to apparent homogeneity. Hydrodynamic studies indicated a relative mass of 88 kDa suggesting a homopentamer structure. The enzyme was shown to catalyze the formation of riboflavin from 6,7-dimethyl-8-ribityllumazine at a rate of 24 nmol mg(-1) min(-1) at 40 degrees C. Divalent metal ions, preferably manganese or magnesium, are required for maximum activity. In contrast to pentameric archaeal type riboflavin synthases, orthologs from plants, fungi and eubacteria are trimeric proteins characterized by an internal sequence repeat with similar folding patterns. In these organisms the reaction is achieved by binding the two substrate molecules in an antiparallel orientation. With the enzyme of M.jannaschii, 13C NMR spectroscopy with 13C-labeled 6,7-dimethyl-8-ribityllumazine samples as substrates showed that the regiochemistry of the dismutation reaction is the same as observed in eubacteria and eukaryotes, however, in a non-pseudo-c2 symmetric environment. Whereas the riboflavin synthases of M.jannaschii and M.thermoautotrophicus are devoid of similarity with those of eubacteria and eukaryotes, they have significant sequence similarity with 6,7-dimethyl-8-ribityllumazine synthases catalyzing the penultimate step of riboflavin biosynthesis. 6,7-Dimethyl-8-ribityllumazine synthase and the archaeal riboflavin synthase appear to have diverged early in the evolution of Archaea from a common ancestor. Some Archaea have eubacterial type riboflavin synthases which may have been acquired by lateral gene transfer.     

Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana

Despite the importance of riboflavin as the direct precursor of the cofactors flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), the physiologically relevant catalyst dephosphorylating the riboflavin biosynthesis pathway intermediate 5‐amino‐6‐ribitylamino‐2,4(1H,3H) pyrimidinedione 5′‐phosphate (ARPP) has not been characterized from any organism. By using as the query sequence a previously identified plastidial FMN hydrolase AtcpFHy1 (At1g79790), belonging to the haloacid dehalogenase (HAD) superfamily, seven candidates for the missing ARPP phosphatase were found, cloned, recombinantly expressed, and purified. Activity screening showed that the enzymes encoded by AtcpFHy1, At4g11570, and At4g25840 catalyze dephosphorylation of ARPP. AtcpFHy1 was renamed AtcpFHy/PyrP1, At4g11570 and At4g25840 were named AtPyrP2 and AtGpp1/PyrP3, respectively. Subcellular localization in planta indicated that AtPyrP2 was localized in plastids and AtGpp1/PyrP3 in mitochondria. Biochemical characterization of AtcpFHy/PyrP1 and AtPyrP2 showed that they have similar K_m values for the substrate ARPP, with AtcpFHy/PyrP1 having higher catalytic efficiency. Screening of 21 phosphorylated substrates showed that AtPyrP2 is specific for ARPP. Molecular weights of AtcpFHy/PyrP1 and AtPyrP2 were estimated at 46 and 72 kDa, suggesting dimers. pH and temperature optima for AtcpFHy/PyrP1 and AtPyrP2 were ~7.0–8.5 and 40–50°C. T‐DNA knockout of AtcpFHy/PyrP1 did not affect the flavin profile of the transgenic plants, whereas silencing of AtPyrP2 decreased accumulation of riboflavin, FMN, and FAD. Our results strongly support AtPyrP2 as the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. The identification of this enzyme closes a long‐standing gap in understanding of the riboflavin biosynthesis in plants.     

Genetic control of riboflavin biosynthesis in Pichia guilliermondii yeasts. The detection of a new regulator gene RIB81

The properties of mutants resistant to 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)-isoalloxazine (MTRY) were studied. The mutants were isolated from a genetic line of Pichia guilliermondii. Several of them were riboflavin overproducers and had derepressed flavinogenesis enzymes (GTP cyclohydrolase, 6.7-dimethyl-8-ribityllumazine synthase) in iron-rich medium. An additional derepression of these enzymes as well as derepression of riboflavin synthase occurred in iron-deficient medium. The characters "riboflavin oversynthesis" and "derepression of enzymes" were recessive in mutants of the 1st class, or dominant in those of the 2nd class. The hybrids of analogue-resistant strains of the 1st class with previously isolated regulatory mutants ribR (novel designation rib80) possessed the wild-type phenotype and were only capable of riboflavin overproduction under iron deficiency. Complementation analysis of the MTRY-resistant mutants showed that vitamin B2 oversynthesis and enzymes' derepression in these mutants are caused by impairment of a novel regulatory gene, RIB81. Thus, riboflavin biosynthesis in P. guilliermondii yeast is regulated at least by two genes of the negative action: RIB80 and RIB81. The meiotic segregants which contained rib80 and rib81 mutations did not show additivity in the action of the above regulatory genes. The hybrids of rib81 mutants with natural nonflavinogenic strain P. guilliermondii NF1453-1 were not capable of riboflavin oversythesis in the iron-rich medium. Apparently, the strain NF1453-1 contains an unaltered gene RIB81.     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

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.     

Synergistic effect of rib80 and hit mutations on riboflavin biosynthesis and iron transport in the yeast Pichia guilliermondii

Mutant strains of the yeast Pichia guilliermondii, carrying both rib80 and hit mutations in a haploid genome, were derived from previously obtained strains with defective rib80 or hit genes, exerting negative control of the riboflavin biosynthesis and iron transport in Pichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of the rib80 and hit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of 55Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Complex structure of Bacillus subtilis RibG: the reduction mechanism during riboflavin biosynthesis

Bacterial RibG is a potent target for antimicrobial agents, because it catalyzes consecutive deamination and reduction steps in the riboflavin biosynthesis. In the N-terminal deaminase domain of Bacillus subtilis RibG, structure-based mutational analyses demonstrated that Glu51 and Lys79 are essential for the deaminase activity. In the C-terminal reductase domain, the complex structure with the substrate at 2.56-A resolution unexpectedly showed a ribitylimino intermediate bound at the active site, and hence suggested that the ribosyl reduction occurs through a Schiff base pathway. Lys151 seems to have evolved to ensure specific recognition of the deaminase product rather than the substrate. Glu290, instead of the previously proposed Asp199, would seem to assist in the proton transfer in the reduction reaction. A detailed comparison reveals that the reductase and the pharmaceutically important enzyme, dihydrofolate reductase involved in the riboflavin and folate biosyntheses, share strong conservation of the core structure, cofactor binding, catalytic mechanism, even the substrate binding architecture.     

Biosynthesis of riboflavin: an unusual riboflavin synthase of Methanobacterium thermoautotrophicum.

Riboflavin synthase was purified by a factor of about 1,500 from cell extract of Methanobacterium thermoautotrophicum. The enzyme had a specific activity of about 2,700 nmol mg(-1) h(-1) at 65 degrees C, which is relatively low compared to those of riboflavin synthases of eubacteria and yeast. Amino acid sequences obtained after proteolytic cleavage had no similarity with known riboflavin synthases. The gene coding for riboflavin synthase (designated ribC) was subsequently cloned by marker rescue with a ribC mutant of Escherichia coli. The ribC gene of M. thermoautotrophicum specifies a protein of 153 amino acid residues. The predicted amino acid sequence agrees with the information gleaned from Edman degradation of the isolated protein and shows 67% identity with the sequence predicted for the unannotated reading frame MJ1184 of Methanococcus jannaschii. The ribC gene is adjacent to a cluster of four genes with similarity to the genes cbiMNQO of Salmonella typhimurium, which form part of the cob operon (this operon contains most of the genes involved in the biosynthesis of vitamin B12). The amino acid sequence predicted by the ribC gene of M. thermoautotrophicum shows no similarity whatsoever to the sequences of riboflavin synthases of eubacteria and yeast. Most notably, the M. thermoautotrophicum protein does not show the internal sequence homology characteristic of eubacterial and yeast riboflavin synthases. The protein of M. thermoautotrophicum can be expressed efficiently in a recombinant E. coli strain. The specific activity of the purified, recombinant protein is 1,900 nmol mg(-1) h(-1) at 65 degrees C. In contrast to riboflavin synthases from eubacteria and fungi, the methanobacterial enzyme has an absolute requirement for magnesium ions. The 5' phosphate of 6,7-dimethyl-8-ribityllumazine does not act as a substrate. The findings suggest that riboflavin synthase has evolved independently in eubacteria and methanobacteria.