SpringerLink
Log in

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

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (United Kingdom)

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • 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 15:2168–2171

    Article  CAS  PubMed  Google Scholar 

  • 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 93:182–195

    Article  CAS  PubMed  Google Scholar 

  • Du L, Yang X, Zhu T, Wang F, **ao X, Park H, Gu Q (2009) Diketopiperazine alkaloids from a deep ocean sediment derived fungus Penicillium sp. Chem Pharm Bull 57:873–876

    Article  CAS  PubMed  Google Scholar 

  • 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 22:4650–4653

    Article  CAS  PubMed  Google Scholar 

  • 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 290:1364–1373

    Article  PubMed  Google Scholar 

  • 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 54:1639–1641

    Article  CAS  PubMed  Google Scholar 

  • Jost M, Zocher G, Tarcz S, Matuschek M, **e 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 132:17849–17858

    Article  CAS  PubMed  Google Scholar 

  • 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 50:1402–1405

    Article  CAS  PubMed  Google Scholar 

  • Li S-M (2010) Prenylated indole derivatives from fungi: structure diversity, biological activities, biosynthesis and chemoenzymatic synthesis. Nat Prod Rep 27:57–78

    Article  PubMed  Google Scholar 

  • Li S-M (2011) Genome mining and biosynthesis of fumitremorgin-type alkaloids in ascomycetes. J Antibiot 64:45–49

    Article  CAS  PubMed  Google Scholar 

  • 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 91:1888–1892

    Article  CAS  Google Scholar 

  • 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 60:3424–3431

    Article  CAS  PubMed  Google Scholar 

  • Liebhold M, **e X, Li S-M (2013) Breaking cyclic dipeptide prenyltransferase regioselectivity by unnatural alkyl donors. Org Lett 15:3062–3065

    Article  CAS  PubMed  Google Scholar 

  • 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 106:14309–14314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Newman DJ, Cragg GM (2012) Natural products as sources of new drugs over the 30 years from 1981 to 2010. J Nat Prod 75:311–335

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 79:7895–7904

    Article  PubMed  Google Scholar 

  • 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 121:11964–11975

    Article  CAS  Google Scholar 

  • Schuller JM, Zocher G, Liebhold M, **e 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 422:87–99

    Article  CAS  PubMed  Google Scholar 

  • 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 14:4770–4773

    Article  CAS  PubMed  Google Scholar 

  • Steffan N, Li S-M (2009) Increasing structure diversity of prenylated diketopiperazine derivatives by using a 4-dimethylallyltryptophan synthase. Arch Microbiol 191:461–466

    Article  CAS  PubMed  Google Scholar 

  • Steffan N, Unsöld IA, Li S-M (2007) Chemoenzymatic synthesis of prenylated indole derivatives by using a 4-dimethylallyltryptophan synthase from Aspergillus fumigatus. Chembiochem 8:1298–1307

    Article  CAS  PubMed  Google Scholar 

  • Unsöld IA, Li S-M (2005) Overproduction, purification and characterization of FgaPT2, a dimethylallyltryptophan synthase from Aspergillus fumigatus. Microbiology 151:1499–1505

    Article  PubMed  Google Scholar 

  • Wallwey C, Li S-M (2011) Ergot alkaloids: structure diversity, biosynthetic gene clusters and functional proof of biosynthetic genes. Nat Prod Rep 28:496–510

    Article  CAS  PubMed  Google Scholar 

  • 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 70:1558–1564

    Article  CAS  PubMed  Google Scholar 

  • Williams RM, Stocking EM, Sanz-Cervera JF (2000) Biosynthesis of prenylated alkaloids derived from tryptophan. Topics Curr Chem 209:97–173

    Article  CAS  Google Scholar 

  • Winkelblech J, Fan A, Li S-M (2015a) Prenyltransferases as key enzymes in primary and secondary metabolism. Appl Microbiol Biotechnol 99:7379–7397

    Article  CAS  PubMed  Google Scholar 

  • Winkelblech J, Liebhold M, Gunera J, **e 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 357:975–986

    Article  CAS  Google Scholar 

  • 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 22:3866–3869

    Article  CAS  PubMed  Google Scholar 

  • Woodside AB, Huang Z, Poulter CD (1988) Trisammonium geranyl diphosphate. Org Synth 66:211–215

    Article  CAS  Google Scholar 

  • 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 99:4213–4223

    Article  CAS  PubMed  Google Scholar 

  • Yamakawa T, Ideue E, Shimokawa J, Fukuyama T (2010) Total synthesis of tryprostatins A and B. Angew Chem Int Ed Engl 49:9262–9265

    Article  CAS  PubMed  Google Scholar 

  • 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 70:3859–3863

    Article  CAS  Google Scholar 

  • Yu X, Li S-M (2012) Prenyltransferases of the dimethylallyltryptophan synthase superfamily. Methods Enzymol 516:259–278

    Article  CAS  PubMed  Google Scholar 

  • Yu X, Zocher G, **e 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 20:1492–1501

    Article  CAS  PubMed  Google Scholar 

  • 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 45:1559–1562

    Article  CAS  PubMed  Google Scholar 

  • Zhao L, May JP, Huang J, Perrin DM (2012) Stereoselective synthesis of brevianamide E. Org Lett 14:90–93

    Article  CAS  PubMed  Google Scholar 

  • 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 93:1758–1762

    Article  CAS  Google Scholar 

  • 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 19:17839–17847

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

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

Author information

Authors and Affiliations

  1. Institut für Pharmazeutische Biologie und Biotechnologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037, Marburg, Germany

    Aili Fan & Shu-Ming Li

Authors
  1. Aili Fan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shu-Ming Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shu-Ming Li.

Ethics declarations

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.

Electronic Supplementary Material

ESM 1

(PDF 387 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Fan, A., Li, SM. Saturation mutagenesis on Arg244 of the tryptophan C4-prenyltransferase FgaPT2 leads to enhanced catalytic ability and different preferences for tryptophan-containing cyclic dipeptides. Appl Microbiol Biotechnol 100, 5389–5399 (2016). https://doi.org/10.1007/s00253-016-7365-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-016-7365-3

Keywords

Search

Navigation