1887

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27759-0
2005-05-01
2020-08-07
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/5/mic1511499.html?itemId=/content/journal/micro/10.1099/mic.0.27759-0&mimeType=html&fmt=ahah

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 Biochem72:248–254[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 Biol10:1281–1292[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 Chem256:10917–10923
    [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 Soc126:11432–11433[CrossRef]
    [Google Scholar]
  6. Flieger M., Wurst M., Shelby R. 1997; Ergot alkaloids – sources, structures and analytical methods. Folia Microbiol42:3–29[CrossRef]
    [Google Scholar]
  7. Floss H. G. 1976; Biosynthesis of ergot alkaloids and related compounds. Tetrahedron32:873–912[CrossRef]
    [Google Scholar]
  8. Gebler J. C., Poulter C. D. 1992; Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea. Arch Biochem Biophys296:308–313[CrossRef]
    [Google Scholar]
  9. Gröger D., Floss H. G. 1998; Biochemistry of ergot alkaloids – achievements and challenges. In Alkaloids: Chemistry and Biology vol 50 pp171–218 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. Phytochemistry29:1131–1135[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. Nature227:680–685[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 Biophys177:84–94[CrossRef]
    [Google Scholar]
  14. Liang P. H., Ko T. P., Wang A. H. 2002; Structure, mechanism and function of prenyltransferases. Eur J Biochem269:3339–3354[CrossRef]
    [Google Scholar]
  15. Melzer M., Heide L. 1994; Characterization of polyprenyldiphosphate : 4-hydroxybenzoate polyprenyltransferase from Escherichia coli. Biochim Biophys Acta1212:93–102[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 A100:2316–2321[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 Commun216:119–125[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 Genet261:133–141[CrossRef]
    [Google Scholar]
  21. Tudzynski P., Correia T., Keller U. 2001; Biotechnology and genetics of ergot alkaloids. Appl Microbiol Biotechnol57:593–605[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 Biol41:189–198[CrossRef]
    [Google Scholar]
  23. Williams R. M., Stocking E. M., Sanz-Cervera J. F. 2000; Biosynthesis of prenylated alkaloids derived from tryptophan. Topics Curr Chem209:97–173
    [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 Nenpo21:95–101
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27759-0
Loading
/content/journal/micro/10.1099/mic.0.27759-0
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error