Advertisement

Applied Microbiology and Biotechnology

, Volume 100, Issue 12, pp 5389–5399 | Cite as

Saturation mutagenesis on Arg244 of the tryptophan C4-prenyltransferase FgaPT2 leads to enhanced catalytic ability and different preferences for tryptophan-containing cyclic dipeptides

  • Aili Fan
  • Shu-Ming LiEmail author
Biotechnologically relevant enzymes and proteins

Abstract

FgaPT2 from Aspergillus fumigatus catalyzes a Friedel–Crafts alkylation at C-4 of l-tryptophan and is involved in the biosynthesis of the ergot alkaloids fumigaclavines. Several tryptophan-containing cyclic dipeptides had also been prenylated by FgaPT2, but the turnover rate (k cat) was low. Here, we report the generation of FgaPT2 mutants by saturation mutagenesis at the amino acid residue Arg244 to improve its catalytic efficiency toward cyclic dipeptides. Thirteen mutated enzymes demonstrated up to 76-fold higher turnover number toward seven cyclic dipeptides than the non-mutated FgaPT2. More importantly, the mutated enzymes exhibited different preferences toward these substrates. This study provides a convenient approach for creation of new biocatalysts for production of C4-prenylated cyclic dipeptides.

Keywords

Cyclic dipeptide Dimethylallyltryptophan synthase Enzyme catalysis Friedel–Crafts alkylation Prenyltransferase Saturation mutagenesis 

Notes

Acknowledgments

We thank Lena Ludwig for synthesis of DMAPP, and Nina Zitzer and Stefan Newel for taking MS and NMR spectra, respectively.

Compliance with ethical standards

Funding

This study was funded by Li844/4-1 from the Deutsche Forschungsgemeinschaft. Aili Fan is a recipient of a scholarship from China Scholarship Council (2011601056).

Conflict of interest

The authors declare that they have no conflict of interest.

Human and animal rights

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2016_7365_MOESM1_ESM.pdf (388 kb)
ESM 1 (PDF 387 kb)

References

  1. Cai S, Luan Y, Kong X, Zhu T, Gu Q, Li D (2013) Isolation and photoinduced conversion of 6-epi-stephacidins from Aspergillus taichungensis. Org Lett 15:2168–2171CrossRefPubMedGoogle Scholar
  2. Chen X, Si L, Liu D, Proksch P, Zhang L, Zhou D, Lin W (2015) Neoechinulin B and its analogues as potential entry inhibitors of influenza viruses, targeting viral hemagglutinin. Eur J Med Chem 93:182–195CrossRefPubMedGoogle Scholar
  3. Du L, Yang X, Zhu T, Wang F, Xiao X, Park H, Gu Q (2009) Diketopiperazine alkaloids from a deep ocean sediment derived fungus Penicillium sp. Chem Pharm Bull 57:873–876CrossRefPubMedGoogle Scholar
  4. Du F-Y, Li X-M, Li C-S, Shang Z, Wang B-G (2012) Cristatumins A-D, new indole alkaloids from the marine-derived endophytic fungus Eurotium cristatum EN-220. Bioorg Med Chem Lett 22:4650–4653CrossRefPubMedGoogle Scholar
  5. Fan A, Zocher G, Stec E, Stehle T, Li S-M (2015) Site-directed mutagenesis switching a dimethylallyl tryptophan synthase to a specific tyrosine C3-prenylating enzyme. J Biol Chem 290:1364–1373CrossRefPubMedGoogle Scholar
  6. Itabashi T, Matsuishi N, Hosoe T, Toyazaki N, Udagawa S, Imai T, Adachi M, Kawai K (2006) Two new dioxopiperazine derivatives, arestrictins A and B, isolated from Aspergillus restrictus and Aspergillus penicilloides. Chem Pharm Bull 54:1639–1641CrossRefPubMedGoogle Scholar
  7. Jost M, Zocher G, Tarcz S, Matuschek M, Xie X, Li S-M, Stehle T (2010) Structure-function analysis of an enzymatic prenyl transfer reaction identifies a reaction chamber with modifiable specificity. J Am Chem Soc 132:17849–17858CrossRefPubMedGoogle Scholar
  8. Kuttruff CA, Zipse H, Trauner D (2011) Concise total syntheses of variecolortides A and B through an unusual Hetero-Diels-Alder reaction. Angew Chem Int Ed Engl 50:1402–1405CrossRefPubMedGoogle Scholar
  9. Li S-M (2010) Prenylated indole derivatives from fungi: structure diversity, biological activities, biosynthesis and chemoenzymatic synthesis. Nat Prod Rep 27:57–78CrossRefPubMedGoogle Scholar
  10. Li S-M (2011) Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes. J Antibiot 64:45–49CrossRefPubMedGoogle Scholar
  11. Li D-L, Li X-M, Li T-G, Dang H-Y, Wang B-G (2008) Dioxopiperazine alkaloids produced by the marine mangrove derived endophytic fungus Eurotium rubrum. Helv Chim Acta 91:1888–1892CrossRefGoogle Scholar
  12. Li XJ, Zhang Q, Zhang AL, Gao JM (2012) Metabolites from Aspergillus fumigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J Agric Food Chem 60:3424–3431CrossRefPubMedGoogle Scholar
  13. Liebhold M, Xie X, Li S-M (2013) Breaking cyclic dipeptide prenyltransferase regioselectivity by unnatural alkyl donors. Org Lett 15:3062–3065CrossRefPubMedGoogle Scholar
  14. Metzger U, Schall C, Zocher G, Unsöld I, Stec E, Li S-M, Heide L, Stehle T (2009) The structure of dimethylallyl tryptophan synthase reveals a common architecture of aromatic prenyltransferases in fungi and bacteria. Proc Natl Acad Sci U S A 106:14309–14314CrossRefPubMedPubMedCentralGoogle Scholar
  15. Newman DJ, Cragg GM (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75:311–335CrossRefPubMedPubMedCentralGoogle Scholar
  16. Peng J, Gao H, Li J, Ai J, Geng M, Zhang G, Zhu T, Gu Q, Li D (2014) Prenylated indole diketopiperazines from the marine-derived fungus Aspergillus versicolor. J Org Chem 79:7895–7904CrossRefPubMedGoogle Scholar
  17. Schkeryantz JM, Woo JCG, Siliphaivanh P, Depew KM, Danishefsky SJ (1999) Total synthesis of gypsetin, deoxybrevianamide E, brevianamide E, and tryprostatin B: novel constructions of 2,3-disubstituted indoles. J Am Chem Soc 121:11964–11975CrossRefGoogle Scholar
  18. Schuller JM, Zocher G, Liebhold M, Xie X, Stahl M, Li S-M, Stehle T (2012) Structure and catalytic mechanism of a cyclic dipeptide prenyltransferase with broad substrate promiscuity. J Mol Biol 422:87–99CrossRefPubMedGoogle Scholar
  19. Song F, Liu X, Guo H, Ren B, Chen C, Piggott AM, Yu K, Gao H, Wang Q, Liu M, Liu X, Dai H, Zhang L, Capon RJ (2012) Brevianamides with antitubercular potential from a marine-derived isolate of Aspergillus versicolor. Org Lett 14:4770–4773CrossRefPubMedGoogle Scholar
  20. Steffan N, Li S-M (2009) Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase. Arch Microbiol 191:461–466CrossRefPubMedGoogle Scholar
  21. Steffan N, Unsöld IA, Li S-M (2007) Chemoenzymatic synthesis of prenylated indole derivatives by using a 4-dimethylallyltryptophan synthase from Aspergillus fumigatus. Chembiochem 8:1298–1307CrossRefPubMedGoogle Scholar
  22. Unsöld IA, Li S-M (2005) Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus. Microbiology 151:1499–1505CrossRefPubMedGoogle Scholar
  23. Wallwey C, Li S-M (2011) Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes. Nat Prod Rep 28:496–510CrossRefPubMedGoogle Scholar
  24. Wang WL, Lu ZY, Tao HW, Zhu TJ, Fang YC, Gu QQ, Zhu WM (2007) Isoechinulin-type alkaloids, variecolorins A-L, from halotolerant Aspergillus variecolor. J Nat Prod 70:1558–1564CrossRefPubMedGoogle Scholar
  25. Williams RM, Stocking EM, Sanz-Cervera JF (2000) Biosynthesis of prenylated alkaloids derived from tryptophan. Topics Curr Chem 209:97–173CrossRefGoogle Scholar
  26. Winkelblech J, Fan A, Li S-M (2015a) Prenyltransferases as key enzymes in primary and secondary metabolism. Appl Microbiol Biotechnol 99:7379–7397CrossRefPubMedGoogle Scholar
  27. Winkelblech J, Liebhold M, Gunera J, Xie X, Kolb P, Li S-M (2015b) Tryptophan C5-, C6- and C7-prenylating enzymes displaying a preference for C-6 of the indole ring in the presence of unnatural dimethylallyl diphosphate analogues. Adv Synth Catal 357:975–986CrossRefGoogle Scholar
  28. Wollinsky B, Ludwig L, Hamacher A, Yu X, Kassack MU, Li S-M (2012) Prenylation at the indole ring leads to a significant increase of cytotoxicity of tryptophan-containing cyclic dipeptides. Bioorg Med Chem Lett 22:3866–3869CrossRefPubMedGoogle Scholar
  29. Woodside AB, Huang Z, Poulter CD (1988) Trisammonium geranyl diphosphate. Org Synth 66:211–215CrossRefGoogle Scholar
  30. Wunsch C, Mundt K, Li S-M (2015) Targeted production of secondary metabolites by coexpression of non-ribosomal peptide synthetase and prenyltransferase genes in Aspergillus. Appl Microbiol Biotechnol 99:4213–4223CrossRefPubMedGoogle Scholar
  31. Yamakawa T, Ideue E, Shimokawa J, Fukuyama T (2010) Total synthesis of tryprostatins A and B. Angew Chem Int Ed Engl 49:9262–9265CrossRefPubMedGoogle Scholar
  32. Yang B, Dong J, Lin X, Zhou X, Zhang Y, Liu Y (2014) New prenylated indole alkaloids from fungus Penicillium sp. derived of mangrove soil sample. Tetrahedron 70:3859–3863CrossRefGoogle Scholar
  33. Yu X, Li S-M (2012) Prenyltransferases of the dimethylallyltryptophan synthase superfamily. Methods Enzymol 516:259–278CrossRefPubMedGoogle Scholar
  34. Yu X, Zocher G, Xie X, Liebhold M, Schütz S, Stehle T, Li S-M (2013) Catalytic mechanism of stereospecific formation of cis-configured prenylated pyrroloindoline diketopiperazines by indole prenyltransferases. Chem Biol 20:1492–1501CrossRefPubMedGoogle Scholar
  35. Zhao S, Smith KS, Deveau AM, Dieckhaus CM, Johnson MA, Macdonald TL, Cook JM (2002) Biological activity of the tryprostatins and their diastereomers on human carcinoma cell lines. J Med Chem 45:1559–1562CrossRefPubMedGoogle Scholar
  36. Zhao L, May JP, Huang J, Perrin DM (2012) Stereoselective synthesis of brevianamide E. Org Lett 14:90–93CrossRefPubMedGoogle Scholar
  37. Zhou L-N, Zhu T-J, Cai S-X, Gu Q-Q, Li D-H (2010) Three new indole-containing diketopiperazine alkaloids from a deep-ocean sediment derived fungus Penicillium griseofulvum. Helv Chim Acta 93:1758–1762CrossRefGoogle Scholar
  38. Zou X, Li Y, Zhang X, Li Q, Liu X, Huang Y, Tang T, Zheng S, Wang W, Tang J (2014) A new prenylated indole diketopiperazine alkaloid from Eurotium cristatum. Molecules 19:17839–17847CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  1. 1.Institut für Pharmazeutische Biologie und BiotechnologiePhilipps-Universität MarburgMarburgGermany

Personalised recommendations