Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Free

Abstract

A putative dimethylallyltryptophan synthase gene, , was identified in the genome sequence of . was cloned and overexpressed in . The protein FgaPT2 was purified to near homogeneity and characterized biochemically. This enzyme was found to convert -tryptophan to 4-dimethylallyltryptophan, a reaction known to be the first step in ergot alkaloid biosynthesis. FgaPT2 is a soluble, dimeric protein with a subunit size of 52 kDa, and contains no putative prenyl diphosphate binding site (N/D)DXXD. values for -tryptophan and dimethylallyl diphosphate (DMAPP) were determined as 8 and 4 μM, respectively. Metal ions, such as Mg and Ca, enhance the reaction velocity, but are not essential for the enzymic reaction. FgaPT2 showed a relatively strict substrate specificity for both tryptophan and DMAPP. FgaPT2 is the first enzyme in the biosynthesis of ergot alkaloids to be purified and characterized in homogeneous form after heterologous overproduction.

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2005-05-01
2024-03-28
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References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. D., Seidman J. G., Smith J. A., Struhl K. (editors) 1996 Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  2. Bradford M. M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef]
    [Google Scholar]
  3. Correia T., Grammel N., Ortel I., Keller U., Tudzynski P. 2003; Molecular cloning and analysis of the ergopeptine assembly system in the ergot fungus Claviceps purpurea . Chem Biol 10:1281–1292 [CrossRef]
    [Google Scholar]
  4. Cress W. A., Chayet L. T., Rilling H. C. 1981; Crystallization and partial characterization of dimethylallyl pyrophosphate : l-tryptophan dimethylallyltransferase from Claviceps sp. SD58. J Biol Chem 256:10917–10923
    [Google Scholar]
  5. Edwards D. J., Gerwick W. H. 2004; Lyngbyatoxin biosynthesis: sequence of biosynthetic gene cluster and identification of a novel aromatic prenyltransferase. J Am Chem Soc 126:11432–11433 [CrossRef]
    [Google Scholar]
  6. Flieger M., Wurst M., Shelby R. 1997; Ergot alkaloids – sources, structures and analytical methods. Folia Microbiol 42:3–29 [CrossRef]
    [Google Scholar]
  7. Floss H. G. 1976; Biosynthesis of ergot alkaloids and related compounds. Tetrahedron 32:873–912 [CrossRef]
    [Google Scholar]
  8. Gebler J. C., Poulter C. D. 1992; Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea . Arch Biochem Biophys 296:308–313 [CrossRef]
    [Google Scholar]
  9. Gröger D., Floss H. G. 1998; Biochemistry of ergot alkaloids – achievements and challenges. In Alkaloids: Chemistry and Biology vol 50 pp 171–218 Edited by Cordell G. A. San Diego: Academic Press;
    [Google Scholar]
  10. Hamerski D., Schmitt D., Matern U. 1990; Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures. Phytochemistry 29:1131–1135 [CrossRef]
    [Google Scholar]
  11. Hikawa H., Yokoyama Y., Murakami Y. 2000; A short synthesis of optically active γ,γ-dimethylallyltryptophan (DMAT). Synthesis214–216
    [Google Scholar]
  12. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 [CrossRef]
    [Google Scholar]
  13. Lee S. L., Floss H. G., Heinstein P. 1976; Purification and properties of dimethylallylpyrophosphate: tryptophan dimethylallyl transferase, the first enzyme of ergot alkaloid biosynthesis inClaviceps. sp. SD 58. Arch Biochem Biophys 177:84–94 [CrossRef]
    [Google Scholar]
  14. Liang P. H., Ko T. P., Wang A. H. 2002; Structure, mechanism and function of prenyltransferases. Eur J Biochem 269:3339–3354 [CrossRef]
    [Google Scholar]
  15. Melzer M., Heide L. 1994; Characterization of polyprenyldiphosphate : 4-hydroxybenzoate polyprenyltransferase from Escherichia coli. Biochim Biophys Acta 1212:93–102 [CrossRef]
    [Google Scholar]
  16. Pojer F., Wemakor E., Kammerer B., Chen H., Walsh C. T., Li S.-M., Heide L. 2003; CloQ, a prenyltransferase involved in clorobiocin biosynthesis. Proc Natl Acad Sci U S A 100:2316–2321 [CrossRef]
    [Google Scholar]
  17. Sambrook J., Russell D. W. 2001 Molecular Cloning: a Laboratory Manual Cold Spring Harbor; New York: Cold Spring Harbor Laboratory;
    [Google Scholar]
  18. Spilsbury J. F., Wilkinson S. 1961; Isolation of festuclavine and two new clavine alkaloids from Aspergillus fumigatus. J Chem Soc 2085–2091 [CrossRef]
    [Google Scholar]
  19. Tsai H. F., Wang H., Gebler J. C., Poulter C. D., Schardl C. L. 1995; The Claviceps purpurea gene encoding dimethylallyltryptophan synthase, the committed step for ergot alkaloid biosynthesis. Biochem Biophys Res Commun 216:119–125 [CrossRef]
    [Google Scholar]
  20. Tudzynski P., Holter K., Correia T., Arntz C., Grammel N., Keller U. 1999; Evidence for an ergot alkaloid gene cluster in Claviceps purpurea. Mol Gen Genet 261:133–141 [CrossRef]
    [Google Scholar]
  21. Tudzynski P., Correia T., Keller U. 2001; Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotechnol 57:593–605 [CrossRef]
    [Google Scholar]
  22. Wang J., Machado C., Panaccione D. G., Tsai H. F., Schardl C. L. 2004; The determinant step in ergot alkaloid biosynthesis by an endophyte of perennial ryegrass. Fungal Genet Biol 41:189–198 [CrossRef]
    [Google Scholar]
  23. Williams R. M., Stocking E. M., Sanz-Cervera J. F. 2000; Biosynthesis of prenylated alkaloids derived from tryptophan. Topics Curr Chem 209:97–173
    [Google Scholar]
  24. Yamano T., Kishino K., Yamatodani S., Abe M. 1962; Ergot fungus. XLIX. Investigation on ergot alkaloids found in cultures of Aspergillus fumigatus. Takeda Kenkyusho Nenpo 21:95–101
    [Google Scholar]
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