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Natural Riboflavin Analogs

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Flavins and Flavoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1146))

Abstract

Riboflavin analogs have a good potential to serve as basic structures for the development of novel anti-infectives. Riboflavin analogs have multiple cellular targets, since riboflavin (as a precursor to flavin cofactors) is active at more than one site in the cell. As a result, the frequency of developing resistance to antimicrobials based on riboflavin analogs is expected to be significantly lower. The only known natural riboflavin analog with antibiotic function is roseoflavin from the bacterium Streptomyces davawensis. This antibiotic negatively affects flavoenzymes and FMN riboswitches. Another roseoflavin producer, Streptomyces cinnabarinus, was recently identified. Possibly, flavin analogs with antibiotic activity are more widespread than anticipated. The same could be true for flavin analogs yet to be discovered, which could constitute tools for cellular chemistry, thus allowing a further extension of the catalytic spectrum of flavoenzymes.

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References

  1. Kurth R, Paust J, Hähnlein W (1996) Vitamins, Chapter 7. In: Ullmann’s Encyclopedia of industrial chemistry. Wiley-VCH, Weinheim, pp 521–530

    Google Scholar 

  2. Bacher A (1991) Riboflavin kinase and FAD synthetase. In: Müller F (ed) Chemistry and biochemistry of flavoenzymes. CRC press, Boca Raton, FL, pp 349–370

    Google Scholar 

  3. Ghisla S, Massey V (1986) New flavins for old: artificial flavins as active site probes of flavoproteins. Biochem J 239:1–12

    PubMed Central  PubMed  CAS  Google Scholar 

  4. Massey V, Hemmerich P (1980) Active-site probes of flavoproteins. Biochem Soc Trans 8:246–257

    PubMed  CAS  Google Scholar 

  5. Eirich LD, Vogels GD, Wolfe RS (1978) Proposed structure for coenzyme F420 from Methanobacterium. Biochemistry 17:4583–4593

    Article  PubMed  CAS  Google Scholar 

  6. Eirich LD, Vogels GD, Wolfe RS (1979) Distribution of coenzyme F420 and properties of its hydrolytic fragments. J Bacteriol 140:20–27

    PubMed Central  PubMed  CAS  Google Scholar 

  7. Bardos TJ (1974) Antimetabolites: molecular design and mode of action. Top Curr Chem 52:63–98

    PubMed  CAS  Google Scholar 

  8. Mack M, Grill S (2006) Riboflavin analogs and inhibitors of riboflavin biosynthesis. Appl Microbiol Biotechnol 71:265–275

    Article  PubMed  CAS  Google Scholar 

  9. French GL (2010) The continuing crisis in antibiotic resistance. Int J Antimicrob Agents 36(Suppl 3):S3–S7

    Article  PubMed  CAS  Google Scholar 

  10. Fischer M, Bacher A (2005) Biosynthesis of flavocoenzymes. Nat Prod Rep 22:324–350

    Article  PubMed  CAS  Google Scholar 

  11. Perkins J, Pero J (2002) Biosynthesis of riboflavin, biotin, folic acid, and cobalamin. In: Sonenshein A, Hoch J, Losick R (eds) Bacillus subtilis and its closest relatives: from genes to cells. ASM Press, Washington DC, pp 271–286

    Google Scholar 

  12. Perkins JB, Pero JG, Sloma A (1990) Riboflavin overproducing strains of Bacillus subtilis. European Patent Application 0 405 730 A1

    Google Scholar 

  13. Nudler E, Mironov AS (2004) The riboswitch control of bacterial metabolism. Trends Biochem Sci 29:11–17

    Article  PubMed  CAS  Google Scholar 

  14. Winkler WC, Breaker RR (2005) Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol 59:487–517

    Article  PubMed  CAS  Google Scholar 

  15. Abbas CA, Sibirny AA (2011) Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers. Microbiol Mol Biol Rev 75:321–360

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  16. García Angulo VA, Bonomi HR, Posadas DM, Serer MI, Torres AG, Zorreguieta Á, Goldbaum FA(2013) Identification and characterization of RibN, a novel family of riboflavin transporters from Rhizobium leguminosarum and other proteobacteria. J Bacteriol 195(20):4611–4619

    Google Scholar 

  17. Vogl C, Grill S, Schilling O, Stulke J, Mack M, Stolz J (2007) Characterization of riboflavin (vitamin B2) transport proteins from Bacillus subtilis and Corynebacterium glutamicum. J Bacteriol 189:7367–7375

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  18. Burgess CM, Slotboom DJ, Geertsma ER, Duurkens RH, Poolman B, van Sinderen D (2006) The riboflavin transporter RibU in Lactococcus lactis: molecular characterization of gene expression and the transport mechanism. J Bacteriol 188:2752–2760

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  19. Duurkens RH, Tol MB, Geertsma ER, Permentier HP, Slotboom DJ (2007) Flavin binding to the high affinity riboflavin transporter RibU. J Biol Chem 282:10380–10386

    Article  PubMed  CAS  Google Scholar 

  20. Vitreschak AG, Rodionov DA, Mironov AA, Gelfand MS (2002) Regulation of riboflavin biosynthesis and transport genes in bacteria by transcriptional and translational attenuation. Nucleic Acids Res 30:3141–3151

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  21. Eitinger T, Rodionov DA, Grote M, Schneider E (2011) Canonical and ECF-type ATP-binding cassette importers in prokaryotes: diversity in modular organization and cellular functions. FEMS Microbiol Rev 35:3–67

    Article  PubMed  CAS  Google Scholar 

  22. ter Beek J, Duurkens RH, Erkens GB, Slotboom DJ (2011) Quaternary structure and functional unit of energy coupling factor (ECF)-type transporters. J Biol Chem 286:5471–5475

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  23. Hemberger S, Pedrolli DB, Stolz J, Vogl C, Lehmann M, Mack M (2011) RibM from Streptomyces davawensis is a riboflavin/roseoflavin transporter and may be useful for the optimization of riboflavin production strains. BMC Biotechnol 11:119–129

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  24. Reihl P, Stolz J (2005) The monocarboxylate transporter homolog Mch5p catalyzes riboflavin (vitamin B2) uptake in Saccharomyces cerevisiae. J Biol Chem 280:39809–39817

    Article  PubMed  CAS  Google Scholar 

  25. Yao Y, Yonezawa A, Yoshimatsu H, Masuda S, Katsura T, Inui K (2010) Identification and comparative functional characterization of a new human riboflavin transporter hRFT3 expressed in the brain. J Nutr 140:1220–1226

    Article  PubMed  CAS  Google Scholar 

  26. Yonezawa A, Masuda S, Katsura T, Inui K (2008) Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1. Am J Physiol Cell Physiol 295:C632–C641

    Article  PubMed  CAS  Google Scholar 

  27. Macheroux P, Kappes B, Ealick SE (2011) Flavogenomics—a genomic and structural view of flavin-dependent proteins. FEBS J 278:2625–2634

    Article  PubMed  CAS  Google Scholar 

  28. Langer S, Hashimoto M, Hobl B, Mathes T, Mack M (2013) Flavoproteins are potential targets for the antibiotic roseoflavin in Escherichia coli. J Bacteriol 195(18):4037–4045

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  29. Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299

    Article  PubMed  CAS  Google Scholar 

  30. Mathes T, Vogl C, Stolz J, Hegemann P (2009) In vivo generation of flavoproteins with modified cofactors. J Mol Biol 385:1511–1518

    Article  PubMed  CAS  Google Scholar 

  31. Cowden WB, Butcher GA, Hunt NH, Clark IA, Yoneda F (1987) Antimalarial activity of a riboflavin analog against Plasmodium vinckei in vivo and Plasmodium falciparum in vitro. Am J Trop Med Hyg 37:495–500

    PubMed  CAS  Google Scholar 

  32. Becker K, Christopherson RI, Cowden WB, Hunt NH, Schirmer RH (1990) Flavin analogs with antimalarial activity as glutathione reductase inhibitors. Biochem Pharmacol 39:59–65

    Article  PubMed  CAS  Google Scholar 

  33. DiMarco AA, Bobik TA, Wolfe RS (1990) Unusual coenzymes of methanogenesis. Annu Rev Biochem 59:355–394

    Article  PubMed  CAS  Google Scholar 

  34. White RH (2001) Biosynthesis of the methanogenic cofactors. Vitam Horm 61:299–337

    Article  PubMed  CAS  Google Scholar 

  35. Kuo MS, Yurek DA, Coats JH, Li GP (1989) Isolation and identification of 7,8-didemethyl-8-hydroxy-5-deazariboflavin, an unusual cosynthetic factor in streptomycetes, from Streptomyces lincolnensis. J Antibiot (Tokyo) 42:475–478

    Article  CAS  Google Scholar 

  36. Coats JH, Li GP, Kuo MS, Yurek DA (1989) Discovery, production, and biological assay of an unusual flavenoid cofactor involved in lincomycin biosynthesis. J Antibiot (Tokyo) 42:472–474

    Article  CAS  Google Scholar 

  37. Mao Y, Varoglu M, Sherman DH (1999) Molecular characterization and analysis of the biosynthetic gene cluster for the antitumor antibiotic mitomycin C from Streptomyces lavendulae NRRL 2564. Chem Biol 6:251–263

    Article  PubMed  CAS  Google Scholar 

  38. Daniels L, Bakhiet N, Harmon K (1985) Widespread distribution of a 5-deazaflavin cofactor in actinomycetes and related bacteria. Syst Appl Microbiol 6:12–17

    Article  CAS  Google Scholar 

  39. Purwantini E, Gillis TP, Daniels L (1997) Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic archaea. FEMS Microbiol Lett 146:129–134

    Article  PubMed  CAS  Google Scholar 

  40. Stover CK, Warrener P, VanDevanter DR, Sherman DR, Arain TM, Langhorne MH, Anderson SW, Towell JA, Yuan Y, McMurray DN, Kreiswirth BN, Barry CE, Baker WR (2000) A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 405:962–966

    Article  PubMed  CAS  Google Scholar 

  41. Tachibana S, Murakami T (1975) The isolation and some properties of new flavins (“schizoflavin”) formed by Schizophyllum commune. J Nutr Sci Vitaminol (Tokyo) 21:61–63

    Article  CAS  Google Scholar 

  42. Kisker C, Schindelin H, Rees DC (1997) Molybdenum-cofactor-containing enzymes: structure and mechanism. Annu Rev Biochem 66:233–267

    Article  PubMed  CAS  Google Scholar 

  43. Leimkuhler S, Wuebbens MM, Rajagopalan KV (2011) The history of the discovery of the molybdenum cofactor and novel aspects of its biosynthesis in bacteria. Coord Chem Rev 255:1129–1144

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  44. Mayhew SG, Whitfield CD, Ghisla S, Jorns MS (1974) Identification and properties of new flavins in electron-transferring flavoprotein from Peptostreptococcus elsdenii and pig-liver glycolate oxidase. Eur J Biochem 44:579–591

    Article  PubMed  CAS  Google Scholar 

  45. Ghisla S, Mayhew SG (1973) Identification and structure of a novel flavin prosthetic group associated with reduced nicotinamide adenine dinucleotide dehydrogenase from Peptostreptococcus elsdenii. J Biol Chem 248:6568–6570

    PubMed  CAS  Google Scholar 

  46. Ghisla S, Mayhew SG (1976) Identification and properties of 8-hydroxyflavin–adenine dinucleotide in electron-transferring flavoprotein from Peptostreptococcus elsdenii. Eur J Biochem 63:373–390

    Article  PubMed  CAS  Google Scholar 

  47. Matsui K (1965) Nekoflavin, a new flavin compound, in the choroid of cat’s eye. J Biochem 57:201–206

    PubMed  CAS  Google Scholar 

  48. Matsui K, Kasai S (1996) Identification of nekoflavin as 7 alpha-hydroxyriboflavin. J Biochem 119:441–447

    Article  PubMed  CAS  Google Scholar 

  49. Ohkawa H, Ohishi N, Yagi K (1983) New metabolites of riboflavin appear in human urine. J Biol Chem 258:5623–5628

    PubMed  CAS  Google Scholar 

  50. Ohkawa H, Ohishi N, Yagi K (1983) New metabolites of riboflavin appeared in rat urine. Biochem Int 6:239–247

    PubMed  CAS  Google Scholar 

  51. West DW, Owen EC (1969) The urinary excretion of metabolites of riboflavine by man. Br J Nutr 23:889–898

    Article  PubMed  CAS  Google Scholar 

  52. Susin S, Abian J, Sanchez-Baeza F, Peleato ML, Abadia A, Gelpi E, Abadia J (1993) Riboflavin 3′- and 5′-sulfate, two novel flavins accumulating in the roots of iron-deficient sugar beet (Beta vulgaris). J Biol Chem 268:20958–20965

    PubMed  CAS  Google Scholar 

  53. Otani S, Takatsu M, Nakano M, Kasai S, Miura R (1974) Letter: roseoflavin, a new antimicrobial pigment from Streptomyces. J Antibiot (Tokyo) 27:88–89

    Article  CAS  Google Scholar 

  54. Grill S, Yamaguchi H, Wagner H, Zwahlen L, Kusch U, Mack M (2007) Identification and characterization of two Streptomyces davawensis riboflavin biosynthesis gene clusters. Arch Microbiol 188:377–387

    Article  PubMed  CAS  Google Scholar 

  55. Mansjo M, Johansson J (2011) The riboflavin analog roseoflavin targets an FMN-riboswitch and blocks Listeria monocytogenes growth, but also stimulates virulence gene-expression and infection. RNA Biol 8:674–680

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  56. Jankowitsch F, Kuhm C, Kellner R, Kalinowski J, Pelzer S, Macheroux P, Mack M (2011) A novel N, N-8-amino-8-demethyl-D-riboflavin dimethyltransferase (RosA) catalyzing the two terminal steps of roseoflavin biosynthesis in Streptomyces davawensis. J Biol Chem 286:38275–38285

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  57. Jankowitsch F, Schwarz J, Ruckert C, Gust B, Szczepanowski R, Blom J, Pelzer S, Kalinowski J, Mack M (2012) Genome sequence of the bacterium Streptomyces davawensis JCM 4913 and heterologous production of the unique antibiotic roseoflavin. J Bacteriol 194:6818–6827

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  58. Otani S, Matsui K, Kasai S (1997) Chemistry and biochemistry of 8-aminoflavins. Osaka City Med J 43:107–137

    PubMed  CAS  Google Scholar 

  59. Kasai S, Kubo Y, Yamanaka S, Hirota T, Sato H, Tsuzukida Y, Matusi K (1978) Anti-riboflavin activity of 8N-alkyl analogues of roseoflavin in some Gram-positive bacteria. J Nutr Sci Vitaminol (Tokyo) 24:339–350

    Article  CAS  Google Scholar 

  60. Matsui K, Kasai S (1976) Photolysis products of roseoflavin. In: Singer T (ed) Flavins and flavoproteins. Proc. Int. Symp. 5th, 1975. Elsevier, Amsterdam, pp 328–333

    Google Scholar 

  61. Kasai S, Yamanaka S, Wang SC, Matsui K (1979) Anti-riboflavin activity of 8-O-alkyl derivatives of riboflavin in some Gram-positive bacteria. J Nutr Sci Vitaminol (Tokyo) 25:289–298

    Article  CAS  Google Scholar 

  62. Juri N, Kubo Y, Kasai S, Otani S, Kusunose M, Matsui K (1987) Formation of roseoflavin from 8-amino- and 8-methylamino-8-demethyl-D-riboflavin. J Biochem (Tokyo) 101:705–711

    Article  CAS  Google Scholar 

  63. Matsui K, Juri N, Kubo Y, Kasai S (1979) Formation of roseoflavin from guanine through riboflavin. J Biochem (Tokyo) 86:167–175

    CAS  Google Scholar 

  64. Chen H, Yamase H, Murakami K, Chang CW, Zhao L, Zhao Z, Liu HW (2002) Expression, purification, and characterization of two N, N-dimethyltransferases, tylM1 and desVI, involved in the biosynthesis of mycaminose and desosamine. Biochemistry 41:9165–9183

    Article  PubMed  CAS  Google Scholar 

  65. Cooke G, Legrand YM, Rotello VM (2004) Model systems for flavoenzyme activity: an electrochemically tuneable model of roseoflavin. Chem Commun 1088–1089

    Google Scholar 

  66. Hasford J, Rizzo C (1998) Linear free energy substituent effect on flavin redox chemistry. J Am Chem Soc 120:2251–2255

    Article  CAS  Google Scholar 

  67. Pedrolli DB, Nakanishi S, Barile M, Mansurova M, Carmona EC, Lux A, Gärtner W, Mack M (2011) The antibiotics roseoflavin and 8-demethyl-8-amino-riboflavin from Streptomyces davawensis are metabolized by human flavokinase and human FAD synthetase. Biochem Pharmacol 82:1853–1859

    Article  PubMed  CAS  Google Scholar 

  68. Grill S, Busenbender S, Pfeiffer M, Kohler U, Mack M (2008) The bifunctional flavokinase/flavin adenine dinucleotide synthetase from Streptomyces davawensis produces inactive flavin cofactors and is not involved in resistance to the antibiotic roseoflavin. J Bacteriol 190:1546–1553

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  69. Yorita K, Misaki H, Palfey BA, Massey V (2000) On the interpretation of quantitative structure-function activity relationship data for lactate oxidase. Proc Natl Acad Sci U S A 97:2480–2485

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  70. Walsh C, Fisher J, Spencer R, Graham DW, Ashton WT, Brown JE, Brown RD, Rogers EF (1978) Chemical and enzymatic properties of riboflavin analogues. Biochemistry 17:1942–1951

    Article  PubMed  CAS  Google Scholar 

  71. Shinkai S, Kameoka K, Honda N, Ueda K, Manabe O, Lindsey J (1986) Spectral and reactivity studies of roseoflavin analogs: correlation between reactivity and spectral parameters. Bioorg Chem 14:119–133

    Article  CAS  Google Scholar 

  72. Otani S, Kasai S, Matsui K (1980) Isolation, chemical synthesis, and properties of roseoflavin. Methods Enzymol 66:235–241

    Article  PubMed  CAS  Google Scholar 

  73. Nakanishi M, Yatome C, Ishida N, Kitade Y (2001) Putative ACP phosphodiesterase gene (acpD) encodes an azoreductase. J Biol Chem 276:46394–46399

    Article  PubMed  CAS  Google Scholar 

  74. Langer S, Nakanishi S, Mathes T, Knaus T, Binter A, Macheroux P, Mase T, Miyakawa T, Tanokura M, Mack M (2013) The flavoenzyme azobenzene reductase AzoR from Escherichia coli binds roseoflavin mononucleotide (RoFMN) with high affinity and is less active in its RoFMN form. Biochemistry 52:4288–4295

    Article  PubMed  CAS  Google Scholar 

  75. Ito K, Nakanishi M, Lee WC, Sasaki H, Zenno S, Saigo K, Kitade Y, Tanokura M (2006) Three-dimensional structure of AzoR from Escherichia coli. An oxidereductase conserved in microorganisms. J Biol Chem 281:20567–20576

    Article  PubMed  CAS  Google Scholar 

  76. Caldwell ST, Farrugia LJ, Hewage SG, Kryvokhyzha N, Rotello VM, Cooke G (2009) Model systems for flavoenzyme activity: an investigation of the role functionality attached to the C(7) position of the flavin unit has on redox and molecular recognition properties. Chem Commun 1350–1352

    Google Scholar 

  77. Reddick JJ, Saha S, Lee J, Melnick JS, Perkins J, Begley TP (2001) The mechanism of action of bacimethrin, a naturally occurring thiamin antimetabolite. Bioorg Med Chem Lett 11:2245–2248

    Article  PubMed  CAS  Google Scholar 

  78. Fiehe K, Arenz A, Drewke C, Hemscheidt T, Williamson RT, Leistner E (2000) Biosynthesis of 4′-O-methylpyridoxine (Ginkgotoxin) from primary precursors. J Nat Prod 63:185–189

    Article  PubMed  CAS  Google Scholar 

  79. Blount KF, Breaker RR (2006) Riboswitches as antibacterial drug targets. Nat Biotechnol 24:1558–1564

    Article  PubMed  CAS  Google Scholar 

  80. Ott E, Stolz J, Lehmann M, Mack M (2009) The RFN riboswitch of Bacillus subtilis is a target for the antibiotic roseoflavin produced by Streptomyces davawensis. RNA Biol 6:276–280

    Article  PubMed  CAS  Google Scholar 

  81. Lee ER, Blount KF, Breaker RR (2009) Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression. RNA Biol 6:187–194

    Article  PubMed  CAS  Google Scholar 

  82. Pedrolli DB, Matern A, Wang J, Ester M, Siedler K, Breaker R, Mack M (2012) A highly specialized flavin mononucleotide riboswitch responds differently to similar ligands and confers roseoflavin resistance to Streptomyces davawensis. Nucleic Acids Res 40:8662–8673

    Article  PubMed Central  PubMed  CAS  Google Scholar 

  83. Schwarz G, Mendel RR, Ribbe MW (2009) Molybdenum cofactors, enzymes and pathways. Nature 460:839–847

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was funded by the German “Federal Ministry of Education and Research” (BMBF) (FKZ 17PNT006) (“Qualifizierungs-/Profilierungsgruppe neue Technologien“) and the research training group NANOKAT (FKZ 0316052A) of the BMBF.

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Pedrolli, D.B., Jankowitsch, F., Schwarz, J., Langer, S., Nakanishi, S., Mack, M. (2014). Natural Riboflavin Analogs. In: Weber, S., Schleicher, E. (eds) Flavins and Flavoproteins. Methods in Molecular Biology, vol 1146. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0452-5_3

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