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Examination of Substrate Specificity of the First Adenylation Domain in mcyA Module Involved in Microcystin Biosynthesis

Year 2020, Volume: 7 Issue: 4, 275 - 285, 15.12.2020
https://doi.org/10.21448/ijsm.715530

Abstract

The cyanotoxin microcystin (MC) is a secondary metabolite, synthesized by nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes. It has many isoforms and the mechanism of its diversity is not well understood. One of the MC synthetase genes, mcyA, codes for the McyA module containing two adenylation (A) domains. The first domain, McyA-A1, generally binds to L-serine (L-ser). Then the N-methyl transferase (NMT) domain converts L-Ser into N-methyldehydroalanine (Mdha), which usually occupies position 7 on the MC molecule. However, various other amino acids (AAs) might also be present at this position. In this study, bioinformatic analyses of selected cyanobacteria were performed to understand whether genetic information in the first adenylation domain of mcyA could explain incorporation of different AAs at position 7 of the MC molecule. Binding pocket signatures of McyA-A1 and putative activated AAs were determined via various bioinformatics tools. Maximum likelihood phylogenetic trees of full length mcyA, mcyA-A1 and 16S rRNA genes were prepared in Mega 6. Phylogenetic analysis of mcyA-A1 nucleotide sequences was in agreement with the predictions of activated AAs by McyA-A1. In comparison with the 16S rRNA and full length mcyA gene trees, mcyA-A1 phylogenetic trees suggested horizontal transfer of the A domain in either Planktothrix agardhii (Gomont) Anagnostidis & Komárek or Planktothrix rubescens (De Candolle ex Gomont) Anagnostidis & Komárek strains. Predictions of activated AAs were generally in agreement with the chemically determined position 7 AAs. However, there were exceptions suggesting the multispecificity of the first A domain of McyA in some cyanobacteria.

References

  • Walsh, C.T., O’Brien, R.V., Khosla, C. (2013). Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed Engl., 52(28), 7098–7124. http://dx.doi.org/10.1002/anie.201208344
  • Dittmann, E., Gugger, M., Sivonen, K., Fewer, D.P. (2015). Natural product biosynthetic diversity and comparative genomics of the cyanobacteria. Trends Microbiol., 23(10), 642–652. http://dx.doi.org/10.1016/j.tim.2015.07.008
  • Meyer, S., Kehr, J.C., Mainz, A., Dehm, D., Petras, D., Süssmuth, R.D., Dittmann, E. (2016). Biochemical dissection of the natural diversification of microcystin provides lessons for synthetic biology of NRPS. Cell Chem Biol., 23(4), 462–471. http://dx.doi.org/10.1016/j.chembiol.2016.03.011
  • Tillett, D., Dittmann, E., Erhard, M., von Döhren, H., Börner, T., Neilan, B.A. (2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide–polyketide synthetase system. Chem Biol., 7(10), 753–764. http://dx.doi.org/10.1016/s1074-5521(00)00021-1
  • Dittmann, E., Fewer, D.P., Neilan, B.A. (2013). Cyanobacterial toxins: biosynthetic routes and evolutionary roots- Review Article. FEMS Microbiol Rev., 37, 23-43. http://dx.doi.org/10.1111/j.1574-6976.2012.12000.x
  • Heck, K., Alvarenga, D.O., Shishido, T.K., Varani, A.M., Dörr, F.A., Pinto, E., Rouhiainen, L., Jokela, J., Sivonen, K., Fiore, M. F. (2018). Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella. Toxicon., 141, 43–50. http://dx.doi.org/10.1016/j.toxicon.2017.10.021
  • Shishido, T.K., Jokela, J., Humisto, A., Suurnäkki, S., Wahlsten, M., Alvarenga, D.O., Sivonen, K., Fewer, D.P. (2019). The biosynthesis of rare Homo-amino acid containing variants of microcystin by a benthic cyanobacterium. Mar Drugs, 17(5), 271. http://dx.doi.org/10.3390/md17050271
  • Christiansen, G., Fastner, J., Erhard, M., Borner, T., Dittmann, E. (2003). Microcystin biosynthesis in Planktothrix: Genes, evolution, and manipulation. J. Bacteriol., 185(2), 564–572. http://dx.doi.org/10.1128/jb.185.2.564-572.2003
  • Rouhiainen, L., Vakkilainen, T., Siemer, B.L., Buikema, W., Haselkorn, R., Sivonen, K. (2004). Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90. Appl Environ Microbiol., 70(2), 686–692. http://dx.doi.org/10.1128/aem.70.2.686-692.2004
  • Mootz, H.D, Schwarzer, D., Marahiel, M.A. (2002). Ways of assembling complex natural products on modular nonribosomal peptide synthetases. Chem BioChem., 3, 490–504. http://dx.doi.org/10.1002/1439-7633(20020603)3:6<490::AID-CBIC490>3.0.CO;2-N
  • Kurmayer, R., Christiansen, G., Gumpenberger, M., Fastner, J. (2005). Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix. Microbiol., 151(5), 1525–1533. http://dx.doi.org/10.1099/mic.0.27779-0
  • Fewer, D.P., Tooming-Klunderud, A., Jokela, J., Wahlsten, M., Rouhiainen, L., Kristensen, T., Rohrlack, T., Jakobsen, K., Sivonen, K. (2008). Natural occurrence of microcystin synthetase deletion mutants capable of producing microcystins in strains of the genus Anabaena (Cyanobacteria). Microbiol., 154(4), 1007–1014. http://dx.doi.org/10.1099/mic.0.2007/016097-0
  • Stachelhaus, T., Mootz, H.D., Marahiel, M.A. (1999). The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol., 6(8), 493–505. http://dx.doi.org/10.1016/s1074-5521(99)80082-9
  • Ansari, M.Z., Yadav, G., Gokhale, R.S., Mohanty, D. (2004). NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res., 1(32), 405-413. http://dx.doi.org/10.1093/nar/gkh359
  • Lautru, S., Challis, L.G. (2004). Substrate recognition by nonribosomal peptide synthetase multi-enzymes. Microbiol., 150(6), 1629–1636. http://dx.doi.org/10.1099/mic.0.26837-0
  • Kudo, F., Miyanaga, A., Eguchi, T. (2019). Structural basis of the nonribosomal codes for nonproteinogenic amino acid selective adenylation enzymes in the biosynthesis of natural products. J. Ind Microbiol Bio., 46, 515-536. http://dx.doi.org/10.1007/s10295-018-2084-7
  • Bouaïcha, N., Miles, C.O., Beach, D.G., Labidi, Z., Djabri, A., Benayache, N.Y., Quang, T.N. (2019). Structural Diversity, Characterization and Toxicology of Microcystins. Toxins, 11(12), 714. http://dx.doi.org/10.3390/toxins11120714
  • Yilmaz, M., Foss, A.J., Miles, C.O., Özen, M., Demir, N., Balcı, M., Beach, D.G. (2019). Comprehensive multi-technique approach reveals the high diversity of microcystins in field collections and an associated isolate of Microcystis aeruginosa from a Turkish lake. Toxicon, 167, 87-100. http://dx.doi.org/10.1016/j.toxicon.2019.06.006
  • Fewer, D.P., Rouhiainen, L., Jokela, J., Wahlsten, M., Laakso, K., Wang, H., Sivonen, K. (2007). Recurrent adenylation domain replacement in the microcystin synthetase gene cluster. BMC Evol Biol., 7(1), 183. http://dx.doi.org/10.1186/1471-2148-7-183
  • Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol., 30(12), 2725-2729. http://dx.doi.org/10.1093/molbev/mst197
  • Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res., 32(5), 1792–1797. http://dx.doi.org/10.1093/nar/gkh340
  • Hasegawa, M., Kishino, H., Yano, T. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol., 22(2), 160-174. http://dx.doi.org/10.1007/bf02101694
  • Nei, M., Kumar, S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press: New York, USA, 332 pp, ISBN-10: 0195135857.
  • Mikalsen, B., Boison, G., Skulberg, O.M., Fastner, J., Davies, W., Gabrielsen, T.M., Rudi, K., Jakobsen, K.S. (2003). Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in microcystis strains. J. Bacteriol., 185(9), 2774-2785. http://dx.doi.org/10.1128/jb.185.9.2774-2785.2003
  • Gehringer, M.M., Adler, L., Roberts, A.A., Moffitt, M.C., Mihali, T.K., Mills, T.J. T., Fieker, C., Neilan, B.A. (2012). Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp. ISME J., 6(10), 1834–1847. http://dx.doi.org/10.1038/ismej.2012.25
  • Tooming-Klunderud, A., Fewer, D.P., Rohrlack, T., Jokela, J., Rouhiainen, L., Sivonen, K., Kristensen, T., Jakobsen, K.S. (2008). Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera. BMC Evol. Biol., 8(1), 256. http://dx.doi.org/10.1186/1471-2148-8-256
  • Puddick, J., Prinsep, M., Wood, S., Kaufononga, S., Cary, S., Hamilton, D. (2014). High levels of structural diversity observed in microcystins from Microcystis CAWBG11 and characterization of six new microcystin congeners. Mar. Drugs, 12(11), 5372-5395. http://dx.doi.org/10.3390/md12115372
  • Challis, G.L., Ravel, J., Townsend, C.A. (2000). Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol., 7(3), 211–224. https://doi.org/10.1016/S1074-5521(00)00091-0
  • Sivonen, K., Carmichael, W.W., Namikoshi, M., Rinehart, K.L., Dahlem, A.M., Niemela, S.I. (1990). Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152. Appl. Environ. Microbiol., 56(9), 2650-2657. http://dx.doi.org/10.1128/AEM.56.9.2650-2657.1990
  • Huang, T., Duan, Y., Zou, Y., Deng, Z., Lin, S.b(2018). NRPS protein MarQ catalyzes flexible adenylation and specific S‑methylation. ACS Chem. Biol., 13(9), 2387-2391. http://dx.doi.org/10.1021/acschembio.8b00364
  • Schaffer, M.L., Otten, L.G. (2009). Substrate flexibility of the adenylation reaction in the Tyrocidine non-ribosomal peptide synthetase. J. Mol. Catal B-Enzym., 59(1-3), 140-144. http://dx.doi.org/10.1016/j.molcatb.2009.02.004

Examination of Substrate Specificity of the First Adenylation Domain in mcyA Module Involved in Microcystin Biosynthesis

Year 2020, Volume: 7 Issue: 4, 275 - 285, 15.12.2020
https://doi.org/10.21448/ijsm.715530

Abstract

The cyanotoxin microcystin (MC) is a secondary metabolite, synthesized by nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) enzymes. It has many isoforms and the mechanism of its diversity is not well understood. One of the MC synthetase genes, mcyA, codes for the McyA module containing two adenylation (A) domains. The first domain, McyA-A1, generally binds to L-serine (L-ser). Then the N-methyl transferase (NMT) domain converts L-Ser into N-methyldehydroalanine (Mdha), which usually occupies position 7 on the MC molecule. However, various other amino acids (AAs) might also be present at this position. In this study, bioinformatic analyses of selected cyanobacteria were performed to understand whether genetic information in the first adenylation domain of mcyA could explain incorporation of different AAs at position 7 of the MC molecule. Binding pocket signatures of McyA-A1 and putative activated AAs were determined via various bioinformatics tools. Maximum likelihood phylogenetic trees of full length mcyA, mcyA-A1 and 16S rRNA genes were prepared in Mega 6. Phylogenetic analysis of mcyA-A1 nucleotide sequences was in agreement with the predictions of activated AAs by McyA-A1. In comparison with the 16S rRNA and full length mcyA gene trees, mcyA-A1 phylogenetic trees suggested horizontal transfer of the A domain in either Planktothrix agardhii (Gomont) Anagnostidis & Komárek or Planktothrix rubescens (De Candolle ex Gomont) Anagnostidis & Komárek strains. Predictions of activated AAs were generally in agreement with the chemically determined position 7 AAs. However, there were exceptions suggesting the multispecificity of the first A domain of McyA in some cyanobacteria.

References

  • Walsh, C.T., O’Brien, R.V., Khosla, C. (2013). Nonproteinogenic amino acid building blocks for nonribosomal peptide and hybrid polyketide scaffolds. Angew Chem Int Ed Engl., 52(28), 7098–7124. http://dx.doi.org/10.1002/anie.201208344
  • Dittmann, E., Gugger, M., Sivonen, K., Fewer, D.P. (2015). Natural product biosynthetic diversity and comparative genomics of the cyanobacteria. Trends Microbiol., 23(10), 642–652. http://dx.doi.org/10.1016/j.tim.2015.07.008
  • Meyer, S., Kehr, J.C., Mainz, A., Dehm, D., Petras, D., Süssmuth, R.D., Dittmann, E. (2016). Biochemical dissection of the natural diversification of microcystin provides lessons for synthetic biology of NRPS. Cell Chem Biol., 23(4), 462–471. http://dx.doi.org/10.1016/j.chembiol.2016.03.011
  • Tillett, D., Dittmann, E., Erhard, M., von Döhren, H., Börner, T., Neilan, B.A. (2000). Structural organization of microcystin biosynthesis in Microcystis aeruginosa PCC7806: an integrated peptide–polyketide synthetase system. Chem Biol., 7(10), 753–764. http://dx.doi.org/10.1016/s1074-5521(00)00021-1
  • Dittmann, E., Fewer, D.P., Neilan, B.A. (2013). Cyanobacterial toxins: biosynthetic routes and evolutionary roots- Review Article. FEMS Microbiol Rev., 37, 23-43. http://dx.doi.org/10.1111/j.1574-6976.2012.12000.x
  • Heck, K., Alvarenga, D.O., Shishido, T.K., Varani, A.M., Dörr, F.A., Pinto, E., Rouhiainen, L., Jokela, J., Sivonen, K., Fiore, M. F. (2018). Biosynthesis of microcystin hepatotoxins in the cyanobacterial genus Fischerella. Toxicon., 141, 43–50. http://dx.doi.org/10.1016/j.toxicon.2017.10.021
  • Shishido, T.K., Jokela, J., Humisto, A., Suurnäkki, S., Wahlsten, M., Alvarenga, D.O., Sivonen, K., Fewer, D.P. (2019). The biosynthesis of rare Homo-amino acid containing variants of microcystin by a benthic cyanobacterium. Mar Drugs, 17(5), 271. http://dx.doi.org/10.3390/md17050271
  • Christiansen, G., Fastner, J., Erhard, M., Borner, T., Dittmann, E. (2003). Microcystin biosynthesis in Planktothrix: Genes, evolution, and manipulation. J. Bacteriol., 185(2), 564–572. http://dx.doi.org/10.1128/jb.185.2.564-572.2003
  • Rouhiainen, L., Vakkilainen, T., Siemer, B.L., Buikema, W., Haselkorn, R., Sivonen, K. (2004). Genes coding for hepatotoxic heptapeptides (microcystins) in the cyanobacterium Anabaena strain 90. Appl Environ Microbiol., 70(2), 686–692. http://dx.doi.org/10.1128/aem.70.2.686-692.2004
  • Mootz, H.D, Schwarzer, D., Marahiel, M.A. (2002). Ways of assembling complex natural products on modular nonribosomal peptide synthetases. Chem BioChem., 3, 490–504. http://dx.doi.org/10.1002/1439-7633(20020603)3:6<490::AID-CBIC490>3.0.CO;2-N
  • Kurmayer, R., Christiansen, G., Gumpenberger, M., Fastner, J. (2005). Genetic identification of microcystin ecotypes in toxic cyanobacteria of the genus Planktothrix. Microbiol., 151(5), 1525–1533. http://dx.doi.org/10.1099/mic.0.27779-0
  • Fewer, D.P., Tooming-Klunderud, A., Jokela, J., Wahlsten, M., Rouhiainen, L., Kristensen, T., Rohrlack, T., Jakobsen, K., Sivonen, K. (2008). Natural occurrence of microcystin synthetase deletion mutants capable of producing microcystins in strains of the genus Anabaena (Cyanobacteria). Microbiol., 154(4), 1007–1014. http://dx.doi.org/10.1099/mic.0.2007/016097-0
  • Stachelhaus, T., Mootz, H.D., Marahiel, M.A. (1999). The specificity-conferring code of adenylation domains in nonribosomal peptide synthetases. Chem Biol., 6(8), 493–505. http://dx.doi.org/10.1016/s1074-5521(99)80082-9
  • Ansari, M.Z., Yadav, G., Gokhale, R.S., Mohanty, D. (2004). NRPS-PKS: a knowledge-based resource for analysis of NRPS/PKS megasynthases. Nucleic Acids Res., 1(32), 405-413. http://dx.doi.org/10.1093/nar/gkh359
  • Lautru, S., Challis, L.G. (2004). Substrate recognition by nonribosomal peptide synthetase multi-enzymes. Microbiol., 150(6), 1629–1636. http://dx.doi.org/10.1099/mic.0.26837-0
  • Kudo, F., Miyanaga, A., Eguchi, T. (2019). Structural basis of the nonribosomal codes for nonproteinogenic amino acid selective adenylation enzymes in the biosynthesis of natural products. J. Ind Microbiol Bio., 46, 515-536. http://dx.doi.org/10.1007/s10295-018-2084-7
  • Bouaïcha, N., Miles, C.O., Beach, D.G., Labidi, Z., Djabri, A., Benayache, N.Y., Quang, T.N. (2019). Structural Diversity, Characterization and Toxicology of Microcystins. Toxins, 11(12), 714. http://dx.doi.org/10.3390/toxins11120714
  • Yilmaz, M., Foss, A.J., Miles, C.O., Özen, M., Demir, N., Balcı, M., Beach, D.G. (2019). Comprehensive multi-technique approach reveals the high diversity of microcystins in field collections and an associated isolate of Microcystis aeruginosa from a Turkish lake. Toxicon, 167, 87-100. http://dx.doi.org/10.1016/j.toxicon.2019.06.006
  • Fewer, D.P., Rouhiainen, L., Jokela, J., Wahlsten, M., Laakso, K., Wang, H., Sivonen, K. (2007). Recurrent adenylation domain replacement in the microcystin synthetase gene cluster. BMC Evol Biol., 7(1), 183. http://dx.doi.org/10.1186/1471-2148-7-183
  • Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S. (2013). MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol., 30(12), 2725-2729. http://dx.doi.org/10.1093/molbev/mst197
  • Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res., 32(5), 1792–1797. http://dx.doi.org/10.1093/nar/gkh340
  • Hasegawa, M., Kishino, H., Yano, T. (1985). Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. J. Mol. Evol., 22(2), 160-174. http://dx.doi.org/10.1007/bf02101694
  • Nei, M., Kumar, S. (2000). Molecular Evolution and Phylogenetics. Oxford University Press: New York, USA, 332 pp, ISBN-10: 0195135857.
  • Mikalsen, B., Boison, G., Skulberg, O.M., Fastner, J., Davies, W., Gabrielsen, T.M., Rudi, K., Jakobsen, K.S. (2003). Natural variation in the microcystin synthetase operon mcyABC and impact on microcystin production in microcystis strains. J. Bacteriol., 185(9), 2774-2785. http://dx.doi.org/10.1128/jb.185.9.2774-2785.2003
  • Gehringer, M.M., Adler, L., Roberts, A.A., Moffitt, M.C., Mihali, T.K., Mills, T.J. T., Fieker, C., Neilan, B.A. (2012). Nodularin, a cyanobacterial toxin, is synthesized in planta by symbiotic Nostoc sp. ISME J., 6(10), 1834–1847. http://dx.doi.org/10.1038/ismej.2012.25
  • Tooming-Klunderud, A., Fewer, D.P., Rohrlack, T., Jokela, J., Rouhiainen, L., Sivonen, K., Kristensen, T., Jakobsen, K.S. (2008). Evidence for positive selection acting on microcystin synthetase adenylation domains in three cyanobacterial genera. BMC Evol. Biol., 8(1), 256. http://dx.doi.org/10.1186/1471-2148-8-256
  • Puddick, J., Prinsep, M., Wood, S., Kaufononga, S., Cary, S., Hamilton, D. (2014). High levels of structural diversity observed in microcystins from Microcystis CAWBG11 and characterization of six new microcystin congeners. Mar. Drugs, 12(11), 5372-5395. http://dx.doi.org/10.3390/md12115372
  • Challis, G.L., Ravel, J., Townsend, C.A. (2000). Predictive, structure-based model of amino acid recognition by nonribosomal peptide synthetase adenylation domains. Chem Biol., 7(3), 211–224. https://doi.org/10.1016/S1074-5521(00)00091-0
  • Sivonen, K., Carmichael, W.W., Namikoshi, M., Rinehart, K.L., Dahlem, A.M., Niemela, S.I. (1990). Isolation and characterization of hepatotoxic microcystin homologs from the filamentous freshwater cyanobacterium Nostoc sp. strain 152. Appl. Environ. Microbiol., 56(9), 2650-2657. http://dx.doi.org/10.1128/AEM.56.9.2650-2657.1990
  • Huang, T., Duan, Y., Zou, Y., Deng, Z., Lin, S.b(2018). NRPS protein MarQ catalyzes flexible adenylation and specific S‑methylation. ACS Chem. Biol., 13(9), 2387-2391. http://dx.doi.org/10.1021/acschembio.8b00364
  • Schaffer, M.L., Otten, L.G. (2009). Substrate flexibility of the adenylation reaction in the Tyrocidine non-ribosomal peptide synthetase. J. Mol. Catal B-Enzym., 59(1-3), 140-144. http://dx.doi.org/10.1016/j.molcatb.2009.02.004
There are 31 citations in total.

Details

Primary Language English
Subjects Structural Biology
Journal Section Articles
Authors

Gözde Yaman 0000-0001-6044-3194

Mete Yılmaz 0000-0002-0982-727X

Publication Date December 15, 2020
Submission Date April 6, 2020
Published in Issue Year 2020 Volume: 7 Issue: 4

Cite

APA Yaman, G., & Yılmaz, M. (2020). Examination of Substrate Specificity of the First Adenylation Domain in mcyA Module Involved in Microcystin Biosynthesis. International Journal of Secondary Metabolite, 7(4), 275-285. https://doi.org/10.21448/ijsm.715530
International Journal of Secondary Metabolite

e-ISSN: 2148-6905