1887

Abstract

We characterized the biosynthetic pathway of tetrahydrobiopterin (BH) in the lipid-producing fungus . The BH cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H-NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H-NTP to 6-pyruvoyltetrahydropterin (PPH), and sepiapterin reductase (SR) for the conversion of PPH to BH, were expressed heterologously in . The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH in are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH biosynthesis pathway in a fungus.

Funding
This study was supported by the:
  • National Science Foundation of China
  • NSFC (Award 2083600)
  • NSFC (Award 30871952)
  • State Key Laboratory of Food Science and Technology (Award SKLF-MB-200802)
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/content/journal/micro/10.1099/mic.0.051847-0
2011-11-01
2024-12-08
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References

  1. Auerbach G., Herrmann A., Gütlich M., Fischer M., Jacob U., Bacher A., Huber R. ( 1997). The 1.25 A� crystal structure of sepiapterin reductase reveals its binding mode to pterins and brain neurotransmitters. EMBO J 16:7219–7230 [View Article][PubMed]
    [Google Scholar]
  2. Ben J., Lim T. M., Phang V. P., Chan W. K. ( 2003). Cloning and tissue expression of 6-pyruvoyl tetrahydropterin synthase and xanthine dehydrogenase from Poecilia reticulata . Mar Biotechnol (NY) 5:568–578 [View Article][PubMed]
    [Google Scholar]
  3. Bligh E. G., Dyer W. J. ( 1959). A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917 [View Article][PubMed]
    [Google Scholar]
  4. Cha K. W., Jacobson K. B., Yim J. J. ( 1991). Isolation and characterization of GTP cyclohydrolase I from mouse liver. Comparison of normal and the hph-1 mutant. J Biol Chem 266:12294–12300[PubMed]
    [Google Scholar]
  5. Chaney A. L., Marbach E. P. ( 1962). Modified reagents for determination of urea and ammonia. Clin Chem 8:130–132[PubMed]
    [Google Scholar]
  6. Chang A., Scheer M., Grote A., Schomburg I., Schomburg D. ( 2009). brenda, amenda and frenda the enzyme information system: new content and tools in 2009. Nucleic Acids Res 37:Database issueD588–D592 [View Article][PubMed]
    [Google Scholar]
  7. Cho S. H., Na J. U., Youn H., Hwang C. S., Lee C. H., Kang S. O. ( 1999). Sepiapterin reductase producing l-threo-dihydrobiopterin from Chlorobium tepidum . Biochem J 340:497–503 [View Article][PubMed]
    [Google Scholar]
  8. Choi Y. K., Jun S. R., Cha E. Y., Park J. S., Park Y. S. ( 2005). Sepiapterin reductases from Chlorobium tepidum and Chlorobium limicola catalyze the synthesis of l-threo-tetrahydrobiopterin from 6-pyruvoyltetrahydropterin. FEMS Microbiol Lett 242:95–99 [View Article][PubMed]
    [Google Scholar]
  9. Citron B. A., Milstien S., Gutierrez J. C., Levine R. A., Yanak B. L., Kaufman S. ( 1990). Isolation and expression of rat liver sepiapterin reductase cDNA. Proc Natl Acad Sci U S A 87:6436–6440 [View Article][PubMed]
    [Google Scholar]
  10. De Saizieu A., Vankan P., van Loon A. P. ( 1995). Enzymic characterization of Bacillus subtilis GTP cyclohydrolase I. Evidence for a chemical dephosphorylation of dihydroneopterin triphosphate. Biochem J 306:371–377[PubMed]
    [Google Scholar]
  11. Du J., Wei N., Xu H., Ge Y., Vásquez-Vivar J., Guan T., Oldham K. T., Pritchard K. A. Jr, Shi Y. ( 2009). Identification and functional characterization of phosphorylation sites on GTP cyclohydrolase I. Arterioscler Thromb Vasc Biol 29:2161–2168 [View Article][PubMed]
    [Google Scholar]
  12. Finn R. D., Mistry J., Schuster-Böckler B., Griffiths-Jones S., Hollich V., Lassmann T., Moxon S., Marshall M., Khanna A. et al. & other authors ( 2006). Pfam: clans, web tools and services. Nucleic Acids Res 34:Database issueD247–D251 [View Article][PubMed]
    [Google Scholar]
  13. Forrest H. S., Van Baalen C. ( 1970). Microbiology of unconjugated pteridines. Annu Rev Microbiol 24:91–108 [View Article][PubMed]
    [Google Scholar]
  14. Hasegawa H., Sawabe K., Nakanishi N., Wakasugi O. K. ( 2005). Delivery of exogenous tetrahydrobiopterin (BH4) to cells of target organs: role of salvage pathway and uptake of its precursor in effective elevation of tissue BH4 . Mol Genet Metab 86:Suppl. 1S2–S10 [View Article][PubMed]
    [Google Scholar]
  15. He A., Rosazza J. P. ( 2003). GTP cyclohydrolase I: purification, characterization, and effects of inhibition on nitric oxide synthase in Nocardia species. Appl Environ Microbiol 69:7507–7513 [View Article][PubMed]
    [Google Scholar]
  16. He A., Simpson D. R., Daniels L., Rosazza J. P. ( 2004). Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I. Protein Expr Purif 35:171–180 [View Article][PubMed]
    [Google Scholar]
  17. Higgins C. E., Gross S. S. ( 2011). The N-terminal peptide of mammalian GTP cyclohydrolase I is an autoinhibitory control element and contributes to binding the allosteric regulatory protein GFRP. J Biol Chem 286:11919–11928 [View Article][PubMed]
    [Google Scholar]
  18. Inoue Y., Kawasaki Y., Harada T., Hatakeyama K., Kagamiyama H. ( 1991). Purification and cDNA cloning of rat 6-pyruvoyl-tetrahydropterin synthase. J Biol Chem 266:20791–20796[PubMed]
    [Google Scholar]
  19. Kanehisa M. ( 1997). A database for post-genome analysis. Trends Genet 13:375–376 [View Article][PubMed]
    [Google Scholar]
  20. Kanehisa M., Goto S., Kawashima S., Okuno Y., Hattori M. ( 2004). The KEGG resource for deciphering the genome. Nucleic Acids Res 32:Database issueD277–D280 [View Article][PubMed]
    [Google Scholar]
  21. Kang D., Kim S., Yim J. ( 1998). Biosynthetic enzymes of tetrahydrolimipterin from green sulfur bacterium Chlorobium limicola . Pteridines 9:69–84 [CrossRef]
    [Google Scholar]
  22. Kaufman S. ( 1967). Pteridine cofactors. Annu Rev Biochem 36:171–184 [View Article][PubMed]
    [Google Scholar]
  23. Kaufman S. ( 1993). New tetrahydrobiopterin-dependent systems. Annu Rev Nutr 13:261–286 [View Article][PubMed]
    [Google Scholar]
  24. Kendrick A., Ratledge C. ( 1992). Desaturation of polyunsaturated fatty acids in Mucor circinelloides and the involvement of a novel membrane-bound malic enzyme. Eur J Biochem 209:667–673 [View Article][PubMed]
    [Google Scholar]
  25. Kim Y. A., Chung H. J., Kim Y. J., Choi Y. K., Hwang Y. K., Lee S. W., Park Y. S. ( 2000). Characterization of recombinant Dictyostelium discoideum sepiapterin reductase expressed in E. coli . Mol Cells 10:405–410[PubMed]
    [Google Scholar]
  26. Lee S. W., Lee H. W., Chung H. J., Kim Y. A., Kim Y. J., Hahn Y., Chung J. H., Park Y. S. ( 1999). Identification of the genes encoding enzymes involved in the early biosynthetic pathway of pteridines in Synechocystis sp. PCC 6803. FEMS Microbiol Lett 176:169–176 [View Article][PubMed]
    [Google Scholar]
  27. Maden B. E. ( 2000). Tetrahydrofolate and tetrahydromethanopterin compared: functionally distinct carriers in C1 metabolism. Biochem J 350:609–629 [View Article][PubMed]
    [Google Scholar]
  28. Maier J., Ninnemann H. ( 1995). Biosynthesis of pteridines in Neurospora crassa, Phycomyces blakesleeanus and Euglena gracilis: detection and characterization of biosynthetic enzymes. Photochem Photobiol 61:43–53 [View Article][PubMed]
    [Google Scholar]
  29. Maier J., Hecker R., Rockel P., Ninnemann H. ( 2001). Role of nitric oxide synthase in the light-induced development of sporangiophores in Phycomyces blakesleeanus . Plant Physiol 126:1323–1330 [View Article][PubMed]
    [Google Scholar]
  30. Maita N., Hatakeyama K., Okada K., Hakoshima T. ( 2004). Structural basis of biopterin-induced inhibition of GTP cyclohydrolase I by GFRP, its feedback regulatory protein. J Biol Chem 279:51534–51540 [View Article][PubMed]
    [Google Scholar]
  31. Milstien S., Jaffe H., Kowlessur D., Bonner T. I. ( 1996). Purification and cloning of the GTP cyclohydrolase I feedback regulatory protein, GFRP. J Biol Chem 271:19743–19751 [View Article][PubMed]
    [Google Scholar]
  32. Nagatsu T., Ichinose H. ( 1999). Regulation of pteridine-requiring enzymes by the cofactor tetrahydrobiopterin. Mol Neurobiol 19:79–96 [View Article][PubMed]
    [Google Scholar]
  33. Ohye T., Hori T. A., Katoh S., Nagatsu T., Ichinose H. ( 1998). Genomic organization and chromosomal localization of the human sepiapterin reductase gene. Biochem Biophys Res Commun 251:597–602 [View Article][PubMed]
    [Google Scholar]
  34. Park Y. S., Kim J. H., Jacobson K. B., Yim J. J. ( 1990). Purification and characterization of 6-pyruvoyl-tetrahydropterin synthase from Drosophila melanogaster . Biochim Biophys Acta 1038:186–194 [View Article][PubMed]
    [Google Scholar]
  35. Ploom T., Thöny B., Yim J., Lee S., Nar H., Leimbacher W., Richardson J., Huber R., Auerbach G. ( 1999). Crystallographic and kinetic investigations on the mechanism of 6-pyruvoyl tetrahydropterin synthase. J Mol Biol 286:851–860 [View Article][PubMed]
    [Google Scholar]
  36. Qi B. X., Fraser T., Mugford S., Dobson G., Sayanova O., Butler J., Napier J. A., Stobart A. K., Lazarus C. M. ( 2004). Production of very long chain polyunsaturated omega-3 and omega-6 fatty acids in plants. Nat Biotechnol 22:739–745 [View Article][PubMed]
    [Google Scholar]
  37. Quevillon E., Silventoinen V., Pillai S., Harte N., Mulder N., Apweiler R., Lopez R. ( 2005). InterProScan: protein domains identifier. Nucleic Acids Res 33:Web Server issueW116–W120 [View Article][PubMed]
    [Google Scholar]
  38. Rudzite V., Jurika E., Baier-Bitterlich G., Widner B., Reibnegger G., Fuchs D. ( 1998). Pteridines and lipid metabolism. Pteridines 9:103–112
    [Google Scholar]
  39. Sakuradani E., Ando A., Ogawa J., Shimizu S. ( 2009). Improved production of various polyunsaturated fatty acids through filamentous fungus Mortierella alpina breeding. Appl Microbiol Biotechnol 84:1–10 [View Article][PubMed]
    [Google Scholar]
  40. Seong C., Kim Y. A., Chung H. J., Park D., Yim J., Baek K., Park Y. S., Han K., Yoon J. ( 1998). Isolation and characterization of the Drosophila melanogaster cDNA encoding the sepiapterin reductase. Biochim Biophys Acta 1443:239–244[PubMed] [CrossRef]
    [Google Scholar]
  41. Shen R. S., Alam A., Zhang Y. X. ( 1989). Human liver GTP cyclohydrolase I: purification and some properties. Biochimie 71:343–349 [View Article][PubMed]
    [Google Scholar]
  42. Son J. K., Rosazza J. P. N. ( 2000). Cyclic guanosine-3′,5′-monophosphate and biopteridine biosynthesis in Nocardia sp. J Bacteriol 182:3644–3648 [View Article][PubMed]
    [Google Scholar]
  43. Stanger O. ( 2002). Physiology of folic acid in health and disease. Curr Drug Metab 3:211–223 [View Article][PubMed]
    [Google Scholar]
  44. Sueoka T., Katoh S. ( 1982). Purification and characterization of sepiapterin reductase from rat erythrocytes. Biochim Biophys Acta 717:265–271[PubMed] [CrossRef]
    [Google Scholar]
  45. Supangat S., Seo K. H., Choi Y. K., Park Y. S., Son D., Han C. D., Lee K. H. ( 2006). Structure of Chlorobium tepidum sepiapterin reductase complex reveals the novel substrate binding mode for stereospecific production of l-threo-tetrahydrobiopterin. J Biol Chem 281:2249–2256 [View Article][PubMed]
    [Google Scholar]
  46. Supangat S., Park S. O., Seo K. H., Lee S. Y., Park Y. S., Lee K. H. ( 2008). Role of Phe-99 and Trp-196 of sepiapterin reductase from Chlorobium tepidum in the production of l-threo-tetrahydrobiopterin. Acta Biochim Biophys Sin (Shanghai) 40:513–518 [View Article][PubMed]
    [Google Scholar]
  47. Tatusov R. L., Fedorova N. D., Jackson J. D., Jacobs A. R., Kiryutin B., Koonin E. V., Krylov D. M., Mazumder R., Mekhedov S. L. et al. & other authors ( 2003). The COG database: an updated version includes eukaryotes. BMC Bioinformatics 4:41 [View Article][PubMed]
    [Google Scholar]
  48. Thöny B., Leimbacher W., Bürgisser D., Heizmann C. W. ( 1992). Human 6-pyruvoyltetrahydropterin synthase: cDNA cloning and heterologous expression of the recombinant enzyme. Biochem Biophys Res Commun 189:1437–1443 [View Article][PubMed]
    [Google Scholar]
  49. Thöny B., Auerbach G., Blau N. ( 2000). Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem J 347:1–16 [View Article][PubMed]
    [Google Scholar]
  50. Vilaseca M. A., Lambruschini N., Gómez-López L., Gutiérrez A., Moreno J., Tondo M., Artuch R., Campistol J. ( 2010). Long-chain polyunsaturated fatty acid status in phenylketonuric patients treated with tetrahydrobiopterin. Clin Biochem 43:411–415 [View Article][PubMed]
    [Google Scholar]
  51. Viveros O. H., Lee C. L., Abou-Donia M. M., Nixon J. C., Nichol C. A. ( 1981). Biopterin cofactor biosynthesis: independent regulation of GTP cyclohydrolase in adrenal medulla and cortex. Science 213:349–350 [View Article][PubMed]
    [Google Scholar]
  52. Weisberg E. P., O’Donnell J. M. ( 1986). Purification and characterization of GTP cyclohydrolase I from Drosophila melanogaster . J Biol Chem 261:1453–1458[PubMed]
    [Google Scholar]
  53. Werner-Felmayer G., Golderer G., Werner E. R. ( 2002). Tetrahydrobiopterin biosynthesis, utilization and pharmacological effects. Curr Drug Metab 3:159–173 [View Article][PubMed]
    [Google Scholar]
  54. White S. W., Zheng J., Zhang Y. M., Rock C. O. ( 2005). The structural biology of type II fatty acid biosynthesis. Annu Rev Biochem 74:791–831 [View Article][PubMed]
    [Google Scholar]
  55. Witter K., Cahill D. J., Werner T., Ziegler I., Rödl W., Bacher A., Gütlich M. ( 1996). Molecular cloning of a cDNA coding for GTP cyclohydrolase I from Dictyostelium discoideum . Biochem J 319:27–32[PubMed]
    [Google Scholar]
  56. Woo H. J., Hwang Y. K., Kim Y. J., Kang J. Y., Choi Y. K., Kim C. G., Park Y. S. ( 2002). Escherichia coli 6-pyruvoyltetrahydropterin synthase ortholog encoded by ygcM has a new catalytic activity for conversion of sepiapterin to 7,8-dihydropterin. FEBS Lett 523:234–238 [View Article][PubMed]
    [Google Scholar]
  57. Wu C. H., Apweiler R., Bairoch A., Natale D. A., Barker W. C., Boeckmann B., Ferro S., Gasteiger E., Huang H. et al. & other authors ( 2006). The Universal Protein Resource (UniProt): an expanding universe of protein information. Nucleic Acids Res 34:Database issueD187–D191 [View Article][PubMed]
    [Google Scholar]
  58. Wynn J. P., bin Abdul Hamid A., Ratledge C. ( 1999). The role of malic enzyme in the regulation of lipid accumulation in filamentous fungi. Microbiology 145:1911–1917 [View Article][PubMed]
    [Google Scholar]
  59. Yim J. J., Brown G. M. ( 1976). Characteristics of guanosine triphosphate cyclohydrolase I purified from Escherichia coli . J Biol Chem 251:5087–5094[PubMed]
    [Google Scholar]
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