Publications

• Characterization of the anthranilate degradation pathway in Geobacillus thermodenitrificans NG80-2.

  Liu X., Dong Y., Li X., Ren Y., Li Y., Wang W., Wang L., Feng L.

  Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH(2)-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the ant ... >> More

  Anthranilate is an important intermediate of tryptophan metabolism. In this study, a hydroxylase system consisting of an FADH(2)-utilizing monooxygenase (GTNG_3160) and an FAD reductase (GTNG_3158), as well as a bifunctional riboflavin kinase/FMN adenylyltransferase (GTNG_3159), encoded in the anthranilate degradation gene cluster in Geobacillus thermodenitrificans NG80-2 were functionally characterized in vitro. GTNG_3159 produces FAD to be reduced by GTNG_3158 and the reduced FAD (FADH(2)) is utilized by GTNG_3160 to convert anthranilate to 3-hydroxyanthranilate (3-HAA), which is further degraded to acetyl-CoA through a meta-cleavage pathway also encoded in the gene cluster. Utilization of this pathway for the degradation of anthranilate and tryptophan by NG80-2 under physiological conditions was confirmed by real-time RT-PCR analysis of representative genes. This is believed to be the first time that the degradation pathway of anthranilate via 3-HAA has been characterized in a bacterium. This pathway is likely to play an important role in the survival of G. thermodenitrificans in the oil reservoir conditions from which strain NG80-2 was isolated. << Less

  Microbiology 156:589-595(2010) [PubMed] [EuropePMC]

  This publication is cited by 5 other entries.

• Archaeal RibL: a new FAD synthetase that is air sensitive.

  Mashhadi Z., Xu H., Grochowski L.L., White R.H.

  FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. ... >> More

  FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL. << Less

  Biochemistry 49:8748-8755(2010) [PubMed] [EuropePMC]

• An FMN hydrolase is fused to a riboflavin kinase homolog in plants.

  Sandoval F.J., Roje S.

  Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella pa ... >> More

  Riboflavin kinases catalyze synthesis of FMN from riboflavin and ATP. These enzymes have to date been cloned from bacteria, yeast, and mammals, but not from plants. Bioinformatic approaches suggested that diverse plant species, including many angiosperms, two gymnosperms, a moss (Physcomitrella patens), and a unicellular green alga (Chlamydomonas reinhardtii), encode proteins that are homologous to riboflavin kinases of yeast and mammals, but contain an N-terminal domain that belongs to the haloacid dehalogenase superfamily of enzymes. The Arabidopsis homolog of these proteins was cloned by RT-PCR, and was shown to have riboflavin kinase and FMN hydrolase activities by characterizing the recombinant enzyme produced in Escherichia coli. Both activities of the purified recombinant Arabidopsis enzyme (AtFMN/FHy) increased when the enzyme assays contained 0.02% Tween 20. The FMN hydrolase activity of AtFMN/FHy greatly decreased when EDTA replaced Mg(2+) in the assays, as expected for a member of the Mg(2+)-dependent haloacid dehalogenase family. The functional overexpression of the individual domains in E. coli establishes that the riboflavin kinase and FMN hydrolase activities reside, respectively, in the C-terminal (AtFMN) and N-terminal (AtFHy) domains of AtFMN/FHy. Biochemical characterization of AtFMN/FHy, AtFMN, and AtFHy shows that the riboflavin kinase and FMN hydrolase domains of AtFMN/FHy can be physically separated, with little change in their kinetic properties. << Less

  J. Biol. Chem. 280:38337-38345(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Identification and characterization of archaeal-type FAD synthase as a novel tractable drug target from the parasitic protozoa <i>Entamoeba histolytica</i>.

  Wulansari D., Jeelani G., Yazaki E., Nozaki T.

  Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). <i>Entamoeba histolytica</ ... >> More

  Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). <i>Entamoeba histolytica</i>, the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from <i>E. histolytica</i>. Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting <i>K</i>_M and <i>k</i>_cat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of <i>EhFADS</i> by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that <i>EhFADS</i> gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in <i>E. histolytica</i>, the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from <i>E. histolytica</i> for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against <i>E. histolytica</i> infections. << Less

  mSphere 9:e0034724-e0034724(2024) [PubMed] [EuropePMC]

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

  Galluccio M., Brizio C., Torchetti E.M., Ferranti P., Gianazza E., Indiveri C., Barile M.

  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 a ... >> More

  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. << Less

  Protein Expr. Purif. 52:175-181(2007) [PubMed] [EuropePMC]

• Uptake and metabolism of antibiotics roseoflavin and 8-demethyl-8-aminoriboflavin in riboflavin-auxotrophic Listeria monocytogenes.

  Matern A., Pedrolli D., Grosshennig S., Johansson J., Mack M.

  The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (v ... >> More

  The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are produced by the bacteria Streptomyces davawensis and Streptomyces cinnabarinus Riboflavin analogs have the potential to be used as broad-spectrum antibiotics, and we therefore studied the metabolism of riboflavin (vitamin B₂), RoF, and AF in the human pathogen Listeria monocytogenes, a bacterium which is a riboflavin auxotroph. We show that the L. monocytogenes protein Lmo1945 is responsible for the uptake of riboflavin, RoF, and AF. Following import, these flavins are phosphorylated/adenylylated by the bifunctional flavokinase/flavin adenine dinucleotide (FAD) synthetase Lmo1329 and adenylylated by the unique FAD synthetase Lmo0728, the first monofunctional FAD synthetase to be described in bacteria. Lmo1329 generates the cofactors flavin mononucleotide (FMN) and FAD, whereas Lmo0728 produces FAD only. The combined activities of Lmo1329 and Lmo0728 are responsible for the intracellular formation of the toxic cofactor analogs roseoflavin mononucleotide (RoFMN), roseoflavin adenine dinucleotide (RoFAD), 8-demethyl-8-aminoriboflavin mononucleotide (AFMN), and 8-demethyl-8-aminoriboflavin adenine dinucleotide (AFAD). In vivo reporter gene assays and in vitro transcription/translation experiments show that the L. monocytogenes FMN riboswitch Rli96, which controls expression of the riboflavin transport gene lmo1945, is negatively affected by riboflavin/FMN and RoF/RoFMN but not by AF/AFMN. Treatment of L. monocytogenes with RoF or AF leads to drastically reduced FMN/FAD levels. We suggest that the reduced flavin cofactor levels in combination with concomitant synthesis of inactive cofactor analogs (RoFMN, RoFAD, AFMN, and AFAD) explain why RoF and AF contribute to antibiotic activity in L. monocytogenes IMPORTANCE: The riboflavin analogs roseoflavin (RoF) and 8-demethyl-8-aminoriboflavin (AF) are small molecules which are produced by Streptomyces davawensis and Streptomyces cinnabarinus RoF and AF were reported to have antibacterial activity, and we studied how these compounds are metabolized by the human bacterial pathogen Listeria monocytogenes We found that the L. monocytogenes protein Lmo1945 mediates uptake of AF and RoF and that the combined activities of the enzymes Lmo1329 and Lmo0728 are responsible for the conversion of AF and RoF to toxic cofactor analogs. Comparative studies with RoF and AF (a weaker antibiotic) suggest that the reduction in FMN/FAD levels and the formation of inactive FMN/FAD analogs explain to a large extent the antibiotic activity of AF and RoF. << Less

  J. Bacteriol. 198:3233-3243(2016) [PubMed] [EuropePMC]

• Purification and characterization of FAD synthetase from Brevibacterium ammoniagenes.

  Manstein D.J., Pai E.F.

  The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although prev ... >> More

  The bifunctional enzyme FAD synthetase from Brevibacterium ammoniagenes was purified by a method involving ATP-affinity chromatography. The final preparation was more than 95% pure. The apparent molecular weight of the enzyme was determined as 38,000 and the isoelectric point as 4.6. Although previous attempts to separate the enzymatic activities had failed, ATP:riboflavin 5'-phosphotransferase and ATP:FMN-adenylyltransferase activities in B. ammoniagenes were believed to be located on two separate proteins with similar properties, possibly joined in a complex. The following evidence, however, suggests the presence of both activities on a single polypeptide chain. The two activities copurify in the same ratio through the purification scheme as presented. Only a single band could be detected when aliquots from the final purification step were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nondenaturing gel electrophoresis, and isoelectric focusing. Edman degradation of the protein yielded a single N-terminal sequence. << Less

  J. Biol. Chem. 261:16169-16173(1986) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.

  Brizio C., Galluccio M., Wait R., Torchetti E.M., Bafunno V., Accardi R., Gianazza E., Indiveri C., Barile M.

  FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as ... >> More

  FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast. << Less

  Biochem. Biophys. Res. Commun. 344:1008-1016(2006) [PubMed] [EuropePMC]