A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis

Streptomyces davawensis is the only organism known to synthesize the antibiotic roseoflavin, a riboflavin (vitamin B(2)) analog. Roseoflavin is converted to roseoflavin mononucleotide (RoFMN) and roseoflavin adenine dinucleotide in the cytoplasm of target cells. (Ribo-)Flavin mononucleotide (FMN) riboswitches are genetic elements, which in many bacteria control genes responsible for the biosynthesis and transport of riboflavin. Streptomyces davawensis is roseoflavin resistant, and the closely related bacterium Streptomyces coelicolor is roseoflavin sensitive. The two bacteria served as models to investigate roseoflavin resistance of S. davawensis and to analyze the mode of action of roseoflavin in S. coelicolor. Our experiments demonstrate that the ribB FMN riboswitch of S. davawensis (in contrast to the corresponding riboswitch of S. coelicolor) is able to discriminate between the two very similar flavins FMN and RoFMN and shows opposite responses to the latter ligands.     

Regulation of Riboflavin Biosynthesis in Bacillus subtilis Is Affected by the Activity of the Flavokinase/Flavin Adenine Dinucleotide Synthetase Encoded by ribC

This work shows that the ribC wild-type gene product has both flavokinase and flavin adenine dinucleotide synthetase (FAD-synthetase) activities. RibC plays an essential role in the flavin metabolism of Bacillus subtilis, as growth of a ribC deletion mutant strain was dependent on exogenous supply of FMN and the presence of a heterologous FAD-synthetase gene in its chromosome. Upon cultivation with growth-limiting amounts of FMN, this ribC deletion mutant strain overproduced riboflavin, while with elevated amounts of FMN in the culture medium, no riboflavin overproduction was observed. In a B. subtilis ribC820 mutant strain, the corresponding ribC820 gene product has reduced flavokinase/FAD-synthetase activity. In this strain, riboflavin overproduction was also repressed by exogenous FMN but not by riboflavin. Thus, flavin nucleotides, but not riboflavin, have an effector function for regulation of riboflavin biosynthesis in B. subtilis, and RibC seemingly is not directly involved in the riboflavin regulatory system. The mutation ribC820 leads to deregulation of riboflavin biosynthesis in B. subtilis, most likely by preventing the accumulation of the effector molecule FMN or FAD.     

Probable reaction mechanisms of flavokinase and FAD synthetase from rat liver

A steady-state kinetic analysis with evaluation of product inhibition was accomplished with purified rat liver flavokinase and FAD synthetase. For flavokinase, Km values were calculated as approximately 11 microM for riboflavin and 3.7 microM for ATP. Ki values were calculated for FMN as 6 microM against riboflavin and for ZnADP as 120 microM against riboflavin and 23 microM against ZnATP. From the inhibition pattern, the flavokinase reaction followed an ordered bi bi mechanism in which riboflavin binds first followed by ATP; ADP is released first followed by FMN. For FAD synthetase, Km values were calculated as 9.1 microM for FMN and 71 microM for MgATP. Ki values were calculated for FAD as 0.75 microM against FMN and 1.3 microM against MgATP and for pyrophosphate as 66 microM against FMN. The product inhibition pattern suggests the FAD synthetase reaction also followed an ordered bi bi mechanism in which ATP binds to enzyme prior to FMN, and pyrophosphate is released from enzyme before FAD. Comparison of Ki values with physiological concentrations of FMN and FAD suggests that the biosynthesis of FAD is most likely regulated by this coenzyme as product at the stage of the FAD synthetase reaction.     

Flavokinase and FAD synthetase from Bacillus subtilis specific for reduced flavins.

A flavokinase preparation from Bacillus subtilis is described which catalyzes the phosphorylation of reduced, but not oxidized, riboflavin. The enzyme is distinguished from other known flavokinases also in having an unusually low Km for the flavin substrate, 50 to 100 nM. ATP is the obligatory phosphate donor; one ATP is utilized for each FMNH2 formed. Mg2+ or Zn2+ is required for the reaction; Co2+ and Mn2+ will substitute, but less effectively. The same enzyme preparation catalyzes the synthesis of FADH2 from FMNH2 and ATP, but not the synthesis of FAD from FMN and ATP. FADH2 is also formed from reduced riboflavin, presumably by sequential flavokinase and FAD synthetase action. Zn2+ cannot replace Mg2+ in FADH2 formation. The reverse reaction, formation of FMN from FAD, occurs only with reduced FAD, giving rise to FMNH2, and is dependent on the presence of inorganic pyrophosphate. The enzyme thus appears to be an FADH2 pyrophosphorylase. The two enzymatic activities, flavokinase and FADH2 pyrophosphorylase, although not separated during the purification procedure, are distinguished by differences in metal ion specificity, in concentration dependence for ATP (apparent Km for ATP = 300 microM for FADH2 synthesis and 6.5 microM for flavokinase), and in the inhibitory effects of riboflavin analogues.     

The riboflavin/FAD cycle in rat liver mitochondria.

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin Km and Vmax values were 4.4+/-1.3 microM and 35+/-5 pmol x min(-1) (mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent Km and apparent Vmax values for FMN were 5.4+/-0.9 microM and 22.9+/-1.4 pmol x min(-1) x (mg matrix protein)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.     

Cloning and characterization of FAD1, the structural gene for flavin adenine dinucleotide synthetase of Saccharomyces cerevisiae.

The FAD1 gene of Saccharomyces cerevisiae has been selected from a genomic library on the basis of its ability to partially correct the respiratory defect of pet mutants previously assigned to complementation group G178. Mutants in this group display a reduced level of flavin adenine dinucleotide (FAD) and an increased level of flavin mononucleotide (FMN) in mitochondria. The restoration of respiratory capability by FAD1 is shown to be due to extragenic suppression. FAD1 codes for an essential yeast protein, since disruption of the gene induces a lethal phenotype. The FAD1 product has been inferred to be yeast FAD synthetase, an enzyme that adenylates FMN to FAD. This conclusion is based on the following evidence. S. cerevisiae transformed with FAD1 on a multicopy plasmid displays an increase in FAD synthetase activity. This is also true when the gene is expressed in Escherichia coli. Lastly, the FAD1 product exhibits low but significant primary sequence similarity to sulfate adenyltransferase, which catalyzes a transfer reaction analogous to that of FAD synthetase. The lower mitochondrial concentration of FAD in G178 mutants is proposed to be caused by an inefficient exchange of external FAD for internal FMN. This is supported by the absence of FAD synthetase activity in yeast mitochondria and the presence of both extramitochondrial and mitochondrial riboflavin kinase, the preceding enzyme in the biosynthetic pathway. A lesion in mitochondrial import of FAD would account for the higher concentration of mitochondrial FMN in the mutant if the transport is catalyzed by an exchange carrier. The ability of FAD1 to suppress impaired transport of FAD is explained by mislocalization of the synthetase in cells harboring multiple copies of the gene. This mechanism of suppression is supported by the presence of mitochondrial FAD synthetase activity in S. cerevisiae transformed with FAD1 on a high-copy-number plasmid but not in mitochondrial of a wild-type strain.     

Analytical and preparative high-performance liquid chromatography separation of flavin and flavin analog coenzymes

Reversed-phase high-performance liquid chromatography has been used to separate a number of flavin and flavin analogs at the riboflavin, FMN, and FAD coenzyme level. Analytical methods were developed which enable the facile determination of a particular flavin or mixture of flavins present. These methods also allowed the separation of oxidized from reduced forms of oxygen-stable flavin analogs. Past investigations have utilized enzymatically synthesized FAD analogs with the problem of potential contamination by other levels of the coenzyme or ATP a cosubstrate in the flavokinase/FAD synthetase reaction. Preparative methods show that all the potential reaction products may be separated from one another thereby allowing the rapid purification of these redox coenzyme analogs. To demonstrate the utility of this method, radiolabeled FAD and 1-deazaFAD were prepared and purified.     

Riboflavin 5'-pyrophosphate: a contaminant of commercial FAD, a coenzyme for FAD-dependent oxidases, and an inhibitor of FAD synthetase.

Commercially available preparations of flavin adenine dinucleotide (FAD) have been found to be 94% pure, the remaining 6% being composed of four or five minor contaminants which can be separated from FAD by reverse-phase high-performance liquid chromatography. FAD purified in this manner has been shown to be 100% pure. One of the contaminants has been identified as riboflavin 5'-pyrophosphate (RPP) by spectroscopic and chemical methods of analysis. This compound has been shown to exhibit biological activity as a weak cofactor for two FAD-requiring enzymes. With the apoprotein of porcine D-amino-acid oxidase, values determined for RPP were 8.4 microM for Km and 0.10 for Vmax compared to 0.47 microM and 0.28 (36 U/mg), respectively, for FAD. With fungal glucose apooxidase, values determined for RPP were 474 nM for Km and 0.02 for Vmax and 45 nM and 0.09 (105 U/mg), respectively, for FAD. RPP can also inhibit FAD biosynthesis. For bovine liver FAD synthetase, a Ki value for RPP against FMN was determined to be 9 microM where Km for FMN was 5.5 microM. These studies illustrate the value of riboflavin 5'-pyrophosphate as a flavin analog for use in the study of structure/function relationships within certain flavin-dependent enzymes.     

Formation of roseoflavin from guanine through riboflavin.

A synthesis of roseoflavin by Streptomyces davawensis from guanine through riboflavin was demonstrated. The lines of evidence are (1)incorporations of 14C of [2-and U-14C] guanine and [2-14C] riboflavin into roseoflavin, (2) no incorporation of 14C of [8-14C] guanine into roseoflavin, (3) localizations of 14C in roseoflavin, and (4) a decrease of specific radioactivity of roseoflavin formed from [2-14C]guanine on addition of riboflavin to the culture. The 14C atoms in roseoflavin formed were localized by radioactivity analysis of the NaOH-hydrolysis products, i.e., urea and 1,2-dihydro-6-methyl-7-dimethylamino-2-keto-1-D-ribityl-3-quinox-alinecarboxylic acid (QC), a new substance. These hydrolysis products were identified by the isolation of dixanthylures, decomposition with urease, and from the properties of QC and QC tetraacetate isolated. These finding suggest that the pyrimidine ring of guanine is conserved in the formation of roseoflavin from guanine through riboflavin.     

Complete purification and general characterization of FAD synthetase from rat liver

Flavin adenine dinucleotide synthetase (ATP:FMN adenylyltransferase, EC 2.7.7.2) was purified about 10,000-fold from the high-speed supernatant of rat liver by a sequence of ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex (A-50), chromatofocusing, FMN-agarose affinity, and Sephadex G-200. The specific activity of the purified enzyme was 133 units (nanomoles of FAD formed per min at 37 degrees C)/mg of protein. This preparation was free from contaminating FAD pyrophosphatase. The apparent molecular weight was estimated to be 97,000 by gel filtration on Sephadex G-200. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an apparent subunit molecular weight of 53,000. Hence, the enzyme is a dimer of approximately 100,000. The enzyme was found most active at pH 7.1, requires Mg2+, and is essentially irreversible in the direction of FAD formation. Kinetic analysis gave Km values of 9.6 microM for FMN and 53 microM for ATP.     

A novel N,N-8-amino-8-demethyl-D-riboflavin Dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis

Streptomyces davawensis synthesizes the antibiotic roseoflavin (RoF) (8-dimethylamino-8-demethyl-D-riboflavin). It was postulated that RoF is synthesized from riboflavin via 8-amino- (AF) and 8-methylamino-8-demethyl-D-riboflavin (MAF). In a cell-free extract of S. davawensis, an S-adenosyl methionine-dependent conversion of AF into MAF and RoF was observed. The corresponding N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity was enriched by column chromatography. The final most active fraction still contained at least five different proteins that were analyzed by enzymatic digestion and concomitant de novo sequencing by MS/MS. One of the sequences matched a hypothetical peptide fragment derived from an as yet uncharacterized open reading frame (sda77220) located in the middle of a (putative) gene cluster within the S. davawensis genome. Expression of ORF sda77220 in Escherichia coli revealed that the corresponding gene product had N,N-8-amino-8-demethyl-d-riboflavin dimethyltransferase activity. Inactivation of ORF sda77220 led to a S. davawensis strain that synthesized AF but not MAF or RoF. Accordingly, as the first identified gene of RoF biosynthesis, ORF sda77220 was named rosA. RosA (347 amino acids; 38 kDa) was purified from a recombinant E. coli strain (as a His(6)-tagged protein) and was biochemically characterized (apparent K(m) for AF = 57.7 ± 9.2 μm; apparent K(D) for AF = 10.0 μm; k(cat) = 0.37 ± 0.02 s(-1)). RosA is a unique enzyme and may be useful for a variety of applications.     

Mutational analysis of the ribC gene of Bacillus subtilis

The nucleotide sequence of the ribC gene encoding the synthesis ofbifunctional flavokinase/flavine adenine nucleotide (FAD) synthetase in Bacillus subtilis have been determined in a family of riboflavin-constitutive mutants. Two mutations have been found in the proximal region of the gene, which controls the transferase (FAD synthase) activity. Three point mutations and one double mutation have been found (in addition to the two mutations that were detected earlier) in the distal region of the gene, which controls the flavokinase (flavin mononucleotide (FMN) synthase) activity. On the basis of all data known to date, it has been concluded that the identified mutations affect riboflavin and ATP binding sites. No mutations have been found in the PTAN conserved sequence, which forms the magnesium and ATP common binding site and is identical for organisms of all organizational levels, from bacteria too humans.     

Peroxidase catalysed aerobic degradation of 5,6,7,8-tetrahydrobiopterin at physiological pH

The aerobic degradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(6H)pterin, and this finally rearranges to 7,8-dihydroxanthopterin. Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3H)pterin. The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 microM and kcat of 22.5 s-1. The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer. By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)-7,8-dihydro(6H)biopterin Km = 1.3 microM and kcat = 22.8 s-1; with (6S)-7,8-dihydro(6H)biopterin Km = 13.5 microM and kcat = 51.6 s-1; and with (6RS)-7,8-dihydro(6H)neopterin Km = 19.2 microM and kcat = 116 s-1.     

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.     

A continuous fluorometric assay for flavokinase. Properties of flavokinase from Peptostreptococcus elsdenii.

A continuous fluorometric assay that utilizes apoflavodoxin as a trapping agent for riboflavin 5'-phosphate (FMN) has been developed for flavokinase (ATP:riboflavin 5'-phosphotransferase, EC 2.7.1.26). Use of this assay is illustrated in a procedure for the partial purification of flavokinase from the strict anaerobe Peptostreptococcus elsdenii. The purified enzyme catalyzed the formation of 8.3 nmol FMN - min-1 - mg-1 at 37 degrees C and had apparent Km values for riboflavin and ATP of 10 and 4.7 micronM, respectively. ATP could be replaced by ADP (22% of the rate observed with ATP) but not by GTP. The enzyme also phosphorylated 5-deaza- and 8-bromoriboflavin with activities of 15 and 70%, respectively, of that with riboflavin; it was inactive with iso riboflavin and deoxyriboflavin.     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

Over-expression in Escherichia coli, purification and characterization of isoform 2 of human FAD synthetase

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.     

Cold-active acetogenic bacteria from surficial sediments of perennially ice-covered Lake Fryxell, Antarctica

RibR is a minor cryptic flavokinase (EC 2.7.1.26) of the Gram-positive bacterium Bacillus subtilis with an unknown cellular function. The flavokinase activity appears to be localized to the N-terminal domain of the protein. Using the yeast three-hybrid system, it was shown that RibR specifically interacts in vivo with the nontranslated wild-type leader of the mRNA of the riboflavin biosynthetic operon. This interaction is lost partially when a leader containing known cis-acting deregulatory mutations in the so-called RFN element is tested. The RFN element is a sequence within the rib-leader mRNA reported to serve as a receptor for an FMN-dependent 'riboswitch'. In RibR itself, interaction was localized to the carboxy-terminate part of the protein, a segment of unknown function that does not show similarity to other proteins in the public databases. Analysis of a ribR-defective strain revealed a mild deregulation with respect to flavin (riboflavin, FMN and FAD) biosynthesis. The results indicate that the RNA-binding protein RibR may be involved in the regulation of the rib genes.