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A pyridoxal 5′-phosphate-dependent Mannich cyclase

Nature Catalysis volume 6, pages 476–486 (2023)Cite this article

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Abstract

Pyridoxal 5′-phosphate (PLP)-dependent enzymes catalyse a diverse range of chemical transformations. Despite their extraordinary functional diversity, no PLP-dependent enzyme is known to catalyse Mannich-type reactions, an important carbon–carbon bond-forming reaction in synthetic organic chemistry. Here we report the discovery of a biosynthetic enzyme LolT, a PLP-dependent enzyme catalysing a stereoselective intramolecular Mannich reaction to construct the pyrrolizidine core scaffold in loline alkaloids. Importantly, its versatile catalytic activity is harnessed for stereoselective synthesis of a variety of conformationally constrained α,α-disubstituted α-amino acids, which bear vicinal quaternary–tertiary stereocentres and various aza(bi)cyclic backbones, such as indolizidine, quinolizidine, pyrrolidine and piperidine. Furthermore, crystallographic and mutagenesis analysis and computational studies together provided mechanistic insights and structural basis for understanding LolT’s catalytic activity and stereoselectivity. Overall, this work expands the biocatalytic repertoire of carbon–carbon bond-forming enzymes and increases our knowledge of the catalytic versatility of PLP-dependent enzymes.

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Fig. 1: PLP-dependent Mannichase and loline biosynthetic pathway.
Fig. 2: Identifying LolT as a PLP-dependent Mannich cyclase.
Fig. 3: Biocatalytic synthesis of 1-azabyclic α-amino acids by LolT.
Fig. 4: Structural characterization of LolT.
Fig. 5: Mutagenesis study and proposed catalytic mechanism of LolT.
Fig. 6: LolT-catalysed two-component Mannich cyclization reactions.

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Data availability

The crystal structure data of LolT is available in the Protein Data Bank under entry 8DL5. The small-molecule X-ray structure data were deposited to the Cambridge Crystallographic Data Centre (CCDC). The deposition numbers are CCDC 2182351 for 3 hydrochloride salt, CCDC 2183481 for 5 hydrochloride salt, CCDC 2183482 for 7 hydrochloride salt, CCDC 2190686 for 9 hydrochloride salt and CCDC 2182349 for (Boc)2-14. All other data are available from the corresponding authors upon reasonable request. Correspondence and requests for materials should be addressed to Y.H.

References

  1. Percudani, R. & Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep. 4, 850–854 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Savile, C. K. et al. Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329, 305–309 (2010).

    CAS  PubMed  Google Scholar 

  3. Phillips, R. S., Poteh, P., Krajcovic, D., Miller, K. A. & Hoover, T. R. Crystal structure of d-ornithine/d-lysine decarboxylase, a stereoinverting decarboxylase: implications for substrate specificity and stereospecificity of fold III decarboxylases. Biochemistry 58, 1038–1042 (2019).

    CAS  PubMed  Google Scholar 

  4. de Chiara, C. et al. d-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition. Nat. Chem. Biol. 16, 686–694 (2020).

    PubMed  PubMed Central  Google Scholar 

  5. Li, Q. et al. Deciphering the biosynthetic origin of l-allo-isoleucine. J. Am. Chem. Soc. 138, 408–415 (2016).

    CAS  PubMed  Google Scholar 

  6. Barra, L. et al. β-NAD as a building block in natural product biosynthesis. Nature 600, 754–758 (2021).

    CAS  PubMed  Google Scholar 

  7. Phillips, R. S., Demidkina, T. V. & Faleev, N. G. Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase. Biochim. Biophys. Acta 1647, 167–172 (2003).

    CAS  PubMed  Google Scholar 

  8. Sato, D. & Nozaki, T. Methionine γ-lyase: the unique reaction mechanism, physiological roles, and therapeutic applications against infectious diseases and cancers. IUBMB Life 61, 1019–1028 (2009).

    CAS  PubMed  Google Scholar 

  9. Watkins-Dulaney, E., Straathof, S. & Arnold, F. Tryptophan synthase: biocatalyst extraordinaire. ChemBioChem 22, 5–16 (2021).

    CAS  PubMed  Google Scholar 

  10. Hai, Y., Chen, M., Huang, A. & Tang, Y. Biosynthesis of mycotoxin fusaric acid and application of a PLP-dependent enzyme for chemoenzymatic synthesis of substituted l-pipecolic acids. J. Am. Chem. Soc. 142, 19668–19677 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Cui, Z. et al. Pyridoxal-5′-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis. Nat. Chem. Biol. 16, 904–911 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Seebeck, F. P. & Hilvert, D. Conversion of a PLP-dependent racemase into an aldolase by a single active site mutation. J. Am. Chem. Soc. 125, 10158–10159 (2003).

    CAS  PubMed  Google Scholar 

  13. Alexeev, D. et al. The crystal structure of 8-amino-7-oxononanoate synthase: a bacterial PLP-dependent, acyl-CoA-condensing enzyme. J. Mol. Biol. 284, 401–419 (1998).

    CAS  PubMed  Google Scholar 

  14. Hoffarth, E. R., Rothchild, K. W. & Ryan, K. S. Emergence of oxygen- and pyridoxal phosphate-dependent reactions. FEBS J. 287, 1403–1428 (2020).

    CAS  PubMed  Google Scholar 

  15. Johnson, L. N. Glycogen phosphorylase: control by phosphorylation and allosteric effectors. FASEB J. 6, 2274–2282 (1992).

    CAS  PubMed  Google Scholar 

  16. Eliot, A. C. & Kirsch, J. F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    CAS  PubMed  Google Scholar 

  17. Rocha, J. F., Pina, A. F., Sousa, S. F. & Cerqueira, N. M. F. S. A. PLP-dependent enzymes as important biocatalysts for the pharmaceutical, chemical and food industries: a structural and mechanistic perspective. Catal. Sci. Tech. 9, 4864–4876 (2019).

    CAS  Google Scholar 

  18. Du, Y.-L. & Ryan, K. S. Pyridoxal phosphate-dependent reactions in the biosynthesis of natural products. Nat. Prod. Rep. 36, 430–457 (2019).

    CAS  PubMed  Google Scholar 

  19. Ellis, J. M. et al. Biocatalytic synthesis of non-standard amino acids by a decarboxylative aldol reaction. Nat. Catal. 5, 136–143 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kimura, T., Vassilev, V. P., Shen, G.-J. & Wong, C.-H. Enzymatic synthesis of β-hydroxy-α-amino acids based on recombinant d- and l-threonine aldolases. J. Am. Chem. Soc. 119, 11734–11742 (1997).

    CAS  Google Scholar 

  21. Percudani, R. & Peracchi, A. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC Bioinformatics 10, 273 (2009).

    PubMed  PubMed Central  Google Scholar 

  22. Mannich, C. Eine Synthese von β-Ketonbasen. Arch. Pharm. 255, 261–276 (1917).

    CAS  Google Scholar 

  23. Arend, M., Westermann, B. & Risch, N. Modern variants of the Mannich reaction. Angew. Chem. Int. Ed. 37, 1044–1070 (1998).

  24. Schirch, V. & Szebenyi, D. M. Serine hydroxymethyltransferase revisited. Curr. Opin. Chem. Biol. 9, 482–487 (2005).

    CAS  PubMed  Google Scholar 

  25. Chen, J. et al. Carbonyl catalysis enables a biomimetic asymmetric Mannich reaction. Science 360, 1438–1442 (2018).

    CAS  PubMed  Google Scholar 

  26. Hamed, R. B. et al. Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases. Nat. Chem. 3, 365–371 (2011).

    CAS  PubMed  Google Scholar 

  27. Zetzsche, L. E. & Narayan, A. R. H. Broadening the scope of biocatalytic C–C bond formation. Nat. Rev. Chem. 4, 334–346 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Schmidt, N. G., Eger, E. & Kroutil, W. Building bridges: biocatalytic C–C-bond formation toward multifunctional products. ACS Catal. 6, 4286–4311 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Scott, T. A. & Piel, J. The hidden enzymology of bacterial natural product biosynthesis. Nat. Rev. Chem. 3, 404–425 (2019).

    PubMed  PubMed Central  Google Scholar 

  30. Schardl, C. L., Grossman, R. B., Nagabhyru, P., Faulkner, J. R. & Mallik, U. P. Loline alkaloids: currencies of mutualism. Phytochemistry 68, 980–996 (2007).

    CAS  PubMed  Google Scholar 

  31. Cakmak, M., Mayer, P. & Trauner, D. An efficient synthesis of loline alkaloids. Nat. Chem. 3, 543–545 (2011).

    CAS  PubMed  Google Scholar 

  32. Spiering, M. J., Moon, C. D., Wilkinson, H. H. & Schardl, C. L. Gene clusters for insecticidal loline alkaloids in the grass-endophytic fungus Neotyphodium uncinatum. Genetics 169, 1403–1414 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Pan, J. et al. Installation of the ether bridge of lolines by the iron- and 2-oxoglutarate-dependent oxygenase, LolO: regio- and stereochemistry of sequential hydroxylation and oxacyclization reactions. Biochemistry 57, 2074–2083 (2018).

    CAS  PubMed  Google Scholar 

  34. Pan, J. et al. Evidence for modulation of oxygen rebound rate in control of outcome by iron(II)- and 2-oxoglutarate-dependent oxygenases. J. Am. Chem. Soc. 141, 15153–15165 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang, D. X., Stromberg, A. J., Spiering, M. J. & Schardl, C. L. Coregulated expression of loline alkaloid-biosynthesis genes in Neotyphodium uncinatum cultures. Fungal Genet Biol. 46, 517–530 (2009).

    CAS  PubMed  Google Scholar 

  36. Fleetwood, D. J., Fokin, M., Styles, K. A., Wickramage, A. S. & Saikia, S. Materials and methods for producing alkaloids. US patent WO2019123399A1 (2019).

  37. Faulkner, J. R. et al. On the sequence of bond formation in loline alkaloid biosynthesis. ChemBioChem 7, 1078–1088 (2006).

    CAS  PubMed  Google Scholar 

  38. Houen, G. et al. Substrate specificity of the bovine serum amine oxidase and in situ characterisation of aminoaldehydes by NMR spectroscopy. Bioorg. Med. Chem. 13, 3783–3796 (2005).

    CAS  PubMed  Google Scholar 

  39. Slabu, I., Galman, J. L., Weise, N. J., Lloyd, R. C. & Turner, N. J. Putrescine transaminases for the synthesis of saturated nitrogen heterocycles from polyamines. ChemCatChem 8, 1038–1042 (2016).

    CAS  Google Scholar 

  40. Schneider, G., Käck, H. & Lindqvist, Y. The manifold of vitamin B6 dependent enzymes. Structure 8, R1–R6 (2000).

    CAS  PubMed  Google Scholar 

  41. Taylor, J. L., Price, J. E. & Toney, M. D. Directed evolution of the substrate specificity of dialkylglycine decarboxylase. Biochim. Biophys. Acta 1854, 146–155 (2015).

    CAS  PubMed  Google Scholar 

  42. Komarov, I. V., Grigorenko, A. O., Turov, A. V. & Khilya, V. P. Conformationally rigid cyclic α-amino acids in the design of peptidomimetics, peptide models and biologically active compounds. Russ. Chem. Rev. 73, 785–810 (2004).

    CAS  Google Scholar 

  43. Tanaka, M. Design and synthesis of chiral α,α-disubstituted amino acids and conformational study of their oligopeptides. Chem. Pharm. Bull. 55, 349–358 (2007).

  44. Crossley, S. W. & Shenvi, R. A. A longitudinal study of alkaloid synthesis reveals functional group interconversions as bad actors. Chem. Rev. 115, 9465–9531 (2015).

    CAS  PubMed  Google Scholar 

  45. Michael, J. P. Simple indolizidine and quinolizidine alkaloids. Alkaloids Chem. Biol. 75, 1–498 (2016).

    CAS  PubMed  Google Scholar 

  46. Ramalingam, K., Lee, K. M., Woodard, R. W., Bleecker, A. B. & Kende, H. Stereochemical course of the reaction catalyzed by the pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase. Proc. Natl Acad. Sci. USA 82, 7820–7824 (1985).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Kim, S. & Park, S. Structural changes during cysteine desulfurase CsdA and sulfur acceptor CsdE interactions provide insight into the trans-persulfuration. J. Biol. Chem. 288, 27172–27180 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Holm, L. Dali server: structural unification of protein families. Nucleic Acids Res. 50, W210–W215 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Irani, S. et al. Snapshots of C–S cleavage in Egt2 reveals substrate specificity and reaction mechanism. Cell Chem. Biol. 25, 519–529.e4 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Bell, E. L. et al. Biocatalysis. Nat. Rev. Methods Prim. 1, 46 (2021).

    CAS  Google Scholar 

  51. Peterson, E. A. & Sober, H. A. Preparation of crystalline phosphorylated derivatives of vitamin B6. J. Am. Chem. Soc. 76, 169–175 (1954).

    CAS  Google Scholar 

  52. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    CAS  PubMed  Google Scholar 

  53. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. The PyMOL Molecular Graphics System, v. 2.1 (Schrödinger, 2018).

  58. Grimme, S. Exploration of chemical compound, conformer, and reaction space with meta-dynamics simulations based on tight-binding quantum chemical calculations. J. Chem. Theory Comput. 15, 2847–2862 (2019).

    CAS  PubMed  Google Scholar 

  59. Frisch, M. J. et al. Gaussian 16, revision C.01 (2016).

  60. Luchini, G., Alegre-Requena, J., Funes-Ardoiz, I. & Paton, R. GoodVibes: automated thermochemistry for heterogeneous computational chemistry data [version 1; peer review: 2 approved with reservations]. F1000Research 9, 291 (2020).

    Google Scholar 

  61. Trott, O., Olson & A, J. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading. J. Comput. Chem. 31, 455–461 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Case, D. A. et al. AMBER 2020 (Univ. of California, 2020).

  63. Chovancova, E. et al. CAVER 3.0: a tool for the analysis of transport pathways in dynamic protein structures. PLoS Comput. Biol. 8, e1002708 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Gaudreault, F., Morency, L. P. & Najmanovich, R. J. NRGsuite: a PyMOL plugin to perform docking simulations in real time using FlexAID. Bioinformatics 31, 3856–3858 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank R. B. Grossman and C. L. Schardl for helpful discussions. We acknowledge the BioPACIFIC MIP (NSF Materials Innovation Platform, DMR-1933487) at the University of California, Santa Barbara for access to instrumentation. This work is supported by the start-up funds from the University of California, Santa Barbara and partially supported by a research grant from the ACS GCI Pharmaceutical Roundtable. We thank G. Wu for small-molecule X-ray structure determinations. We thank the beamline staff at SSRL BL9-2 for assistance with remote data collection on protein crystal diffractions. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract number DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The work at Lawrence Livermore National Laboratory was performed under the auspices of the US Department of Energy by Lawrence Livermore National Laboratory under contract number DE-AC52-07NA27344.

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Author notes

  1. These authors contributed equally: Jinmin Gao, Shaonan Liu.

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, CA, USA

    Jinmin Gao, Shaonan Liu, Chen Zhou, Darwin Lara & Yang Hai

  2. Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA, USA

    Yike Zou

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Contributions

Y.H. designed the overall research. J.G. and S.L. performed organic synthesis. S.L. performed isolation and characterization of enzymatic reaction products. J.G. and C.Z. prepared and characterized the mutant activity. J.G., D.L., S.L. and Y.H. studied the substrate scope. J.G. and Y.H. performed protein crystallography experiments. Y.Z. carried out the computation studies. Y.H. wrote the manuscript, with input from all other co-authors.

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Correspondence to Yike Zou or Yang Hai.

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Competing interests

Y.H., J.G. and S.L. are inventors on a patent application (US provisional patent application number 63/367,977) submitted by the University of California, Santa Barbara that covers the usage of LolT for stereoselective synthesis of heterocyclic α,α-disubstituted amino acids. C.Z., D.L. and Y.Z. declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Methods, Notes, Tables 1–27 and Figs. 1–111.

Reporting Summary

Supplementary Data 1

Electronic structure calculation coordinates.

Supplementary Data 2

Crystallographic data for compound 3·2HCl.

Supplementary Data 3

Crystallographic data for compound 5·2HCl

Supplementary Data 4

Crystallographic data for compound 7·2HCl

Supplementary Data 5

Crystallographic data for compound 9·2HCl

Supplementary Data 6

Crystallographic data for compound (Boc)2-14.

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Gao, J., Liu, S., Zhou, C. et al. A pyridoxal 5′-phosphate-dependent Mannich cyclase. Nat Catal 6, 476–486 (2023). https://doi.org/10.1038/s41929-023-00963-y

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