Biotechnology and Bioprocess Engineering

, Volume 24, Issue 1, pp 215–222 | Cite as

Enzymatic Synthesis of D-pipecolic Acid by Engineering the Substrate Specificity of Trypanosoma cruzi Proline Racemase and Its Molecular Docking Study

  • Sungmin Byun
  • Hyun June Park
  • Jeong Chan Joo
  • Yong Hwan KimEmail author
Research Paper


Pipecolic acid is an unnatural amino acid mostly used for pharmaceutical purposes. Pipecolic acid has two types of enantiomers with different roles in the synthesis of drugs. The development of efficient catalytic methods for the production of enantiopure pipecolic acid is currently a crucial topic of research. Few chemo- or biosynthetic methods have been proposed for the synthesis of pure enantiomers; however, enzymatic conversion of the chirality of pipecolic acid has not been demonstrated because no pipecolic acid racemase has been reported yet. In this work, we attempted to engineer pipecolic acid racemase activity into Trypanosoma cruzi proline racemase (TcPRAC) for the enzymatic synthesis of D-pipecolic acid from L-pipecolic acid. For the binding of pipecolic acid (C6 ring) into the active site of TcPRAC, which was optimized for the original substrate proline (C5 ring), four bulky aromatic residues (Phe102, Phe120, Phe220, and Phe 290) of TcPRAC were mutated to smaller hydrophobic residues. Among the mutants, six single-point mutants (F102A, F102I, F102L, F102V, F290L, and F290V) exhibited significant racemase activity against L-pipecolic acid. The most efficient variant, F102V, showed 74% racemization. Molecular docking simulations revealed that lowering the binding energy of L-pipecolic acid to the active site was important for achieving high racemization activity of TcPRAC mutant proteins.


D-pipecolic acid enzymatic synthesis substrate specificity engineering Trypanosoma cruzi proline racemase racemization 


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  1. 1.
    Bornscheuer, U. T. and M. Pohl (2001) Improved biocatalysts by directed evolution and rational protein design. Current Opinion in Chemical Biology 5: 137–143.CrossRefPubMedGoogle Scholar
  2. 2.
    Privett, H. K., G. Kiss, T. M. Lee, R. Blomberg, R. A. Chica, L. M. Thomas, D. Hilvert, K. N. Houk, and S. L. Mayo (2012) Iterative approach to computational enzyme design. Proceedings of the National Academy of Sciences of the United States of America 109: 3790–3795.CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Hedstrom, L. (2002) Serine protease mechanism and specificity. Chemical Reviews 102: 4501–4523.CrossRefPubMedGoogle Scholar
  4. 4.
    Looger, L. L., M. A. Dwyer, J. J. Smith, and H. W. Hellinga (2003) Computational design of receptor and sensor proteins with novel functions. Nature 423: 185–190.CrossRefPubMedGoogle Scholar
  5. 5.
    Perona, J. J. and C. S. Craik (1995) Structural basis of substrate specificity in the serine proteases. Protein Science, 4: 337–360.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Yeon, Y. J., H.-Y. Park, and Y. J. Yoo (2013) Enzymatic reduction of levulinic acid by engineering the substrate specificity of 3-hydroxybutyrate dehydrogenase. Bioresource Technology, 134: 377–380.CrossRefPubMedGoogle Scholar
  7. 7.
    Vranova, V., L. Lojkova, K. Rejsek, and P. Formanek (2013) Significance of the natural occurrence of L-versus D-pipecolic acid: A review. Chirality 25: 823–831.CrossRefPubMedGoogle Scholar
  8. 8.
    Tanaka, H., A. Kuroda, H. Marusawa, H. Hatanaka, T. Kino, T. Goto, M. Hashimoto, and T. Taga (1987) Structure of FK506, a novel immunosuppressant isolated from Streptomyces. Journal of the American Chemical Society 109: 5031–5033.CrossRefGoogle Scholar
  9. 9.
    Adger, B., U. Dyer, G. Hutton, and M. Woods (1996) Stereospecific synthesis of the anaesthetic levobupivacaine. Tetrahedron Letters 37: 6399–6402.CrossRefGoogle Scholar
  10. 10.
    Germann, U. A., D. Shlyakhter, V. S. Mason, R. E. Zelle, J. P. Duffy, V. Galullo, D. M. and J. O. Saunders, J. Boger, and M. W. Harding (1997) Cellular and biochemical characterization of VX-710 as a chemosensitizer: reversal of Pglycoprotein-mediated multidrug resistance in vitro. ANTICANCER DRUGS 8: 125–140.CrossRefPubMedGoogle Scholar
  11. 11.
    Pettibone, D. J., B. V. Clineschmidt, P. S. Anderson, R. M. Freidinger, G. F. Lundell, L. R. Koupal, C. D. Schwartz, J. M. Williamson, M. A. Goetz, O. D. Hensens, J. M. Liesch, and J. P. Springer (1989) A structurally unique, potent, and selective oxytocin antagonist derived from Streptomyces silvensis. ENDOCRINOLOGY 125: 217–222.CrossRefPubMedGoogle Scholar
  12. 12.
    Vezina, C., A. Kudelski, and S. N. Sehgal (1975) Rapamycin (AY–22,989), a new antifungal antibiotic. I. Taxonomy of the producing streptomycete and isolation of the active principle. J. ANTIBIOT. 28: 721–726.CrossRefPubMedGoogle Scholar
  13. 13.
    Boger, D. L., J. H. Chen, and K. W. Saionz (1996) (−)-Sandramycin: Total synthesis and characterization of DNA binding properties. Journal of the American Chemical Society 118: 1629–1644.CrossRefGoogle Scholar
  14. 14.
    Hirota, A., A. Suzuki, K. Aizawa, and S. Tamura (1973) Structure of Cyl-2, a novel cyclotetrapeptide from Cylindrocladium scoparium. Agricultural and Biological Chemistry 37: 955–956.CrossRefGoogle Scholar
  15. 15.
    Darkin-Rattray, S. J., A. M. Gurnett, R. W. Myers, P. M. Dulski, T. M. Crumley, J. J. Allocco, C. Cannova, P. T. Meinke, S. L. Colletti, M. A. Bednarek, S. B. Singh, M. A. Goetz, A. W. Dombrowski, J. D. Polishook, and D. M. Schmatz (1996) Apicidin: A novel antiprotozoal agent that inhibits parasite histone deacetylase. Proceedings of the National Academy of Sciences of the United States of America 93: 13143–13147.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Chang, Y.-F. and E. Adams (1971) Induction of separate catabolic pathways for L-and D-lysine in Pseudomonas putida. Biochemical and Biophysical Research Communications 45: 570–577.CrossRefPubMedGoogle Scholar
  17. 17.
    Revelles, O., M. Espinosa-Urgel, T. Fuhrer, U. Sauer, and J. L. Ramos (2005) Multiple and interconnected pathways for l-lysine catabolism in Pseudomonas putida KT2440. Journal of Bacteriology 187: 7500–7510.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Myers, A. G., J. L. Gleason, T. Yoon, and D. W. Kung (1997) Highly practical methodology for the synthesis of d-and l-α-amino acids, N-protected α-amino acids, and N-methyl-α-amino acids. Journal of the American Chemical Society 119: 656–673.CrossRefGoogle Scholar
  19. 19.
    Agami, C., C. Kadouri-Puchot, and J. C. Kizirian (2000) A new enantioselective synthesis of (2S)-pipecolic acid. Synthetic Communications 30: 2565–2572.CrossRefGoogle Scholar
  20. 20.
    Ohtani, B., S. Tsuru, S. I. Nishimoto, T. Kagiya, and K. Izawa (1990) Photocatalytic one-step syntheses of cyclic imino acids by aqueous semiconductor suspensions. Journal of Organic Chemistry 55: 5551–5553.CrossRefGoogle Scholar
  21. 21.
    Foti, C. J. and D. L. Comins (1995) Synthesis and reactions of. alpha.-(trifluoromethanesulfonyloxy) enecarbamates prepared from N-acyllactams. Journal of Organic Chemistry 60: 2656–2657.CrossRefGoogle Scholar
  22. 22.
    Fernández-García, C. and M. A. McKervey (1995) A short enantioselective synthesis of pipecolic acid. Tetrahedron: Asymmetry 6: 2905–2906.CrossRefGoogle Scholar
  23. 23.
    Nazabadioko, S., R. J. Pérez, R. Brieva, and V. Gotor (1998) Chemoenzymatic synthesis of (S)-2-cyanopiperidine, a key intermediate in the route to (S)-pipecolic acid and 2-substituted piperidine alkaloids. Tetrahedron Asymmetry 9: 1597–1604.CrossRefGoogle Scholar
  24. 24.
    Sánchez-Sancho, F. and B. Herradón (1998) Short syntheses of (S)-pipecolic acid, (R)-coniine, and (S)-δ-coniceine using biocatalytically-generated chiral building blocks. Tetrahedron Asymmetry 9: 1951–1965.CrossRefGoogle Scholar
  25. 25.
    Namwat, W., Y. Kamioka, H. Kinoshita, Y. Yamada, and T. Nihira (2002) Characterization of virginiamycin S biosynthetic genes from Streptomyces virginiae. Gene 286: 283–290.CrossRefPubMedGoogle Scholar
  26. 26.
    Watanabe, L. A., S. Haranaka, B. Jose, M. Yoshida, T. Kato, M. Moriguchi, K. Soda, and N. Nishino (2005) An efficient access to both enantiomers of pipecolic acid. Tetrahedron: Asymmetry 16: 903–908.CrossRefGoogle Scholar
  27. 27.
    Buschiazzo, A., M. Goytia, F. Schaeffer, W. Degrave, W. Shepard, C. Grégoire, N. Chamond, A. Cosson, A. Berneman, N. Coatnoan, P. M. Alzari, and P. Minoprio (2006) Crystal structure, catalytic mechanism, and mitogenic properties of Trypanosoma cruzi proline racemase. Proceedings of the National Academy of Sciences of the United States of America 103: 1705–1710.CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Reina-San-Martı́n, B., A. Cosson, and P. Minoprio (2000) Lymphocyte polyclonal activation: A pitfall for vaccine design against infectious agents. Parasitology Today 16: 62–67.CrossRefPubMedGoogle Scholar
  29. 29.
    Chamond, N., A. Cosson, N. Coatnoan, and P. Minoprio (2009) Proline racemases are conserved mitogens: Characterization of a Trypanosoma vivax proline racemase. Molecular and Biochemical Parasitology 165: 170–179.CrossRefPubMedGoogle Scholar
  30. 30.
    Reina-San-Martín, B., W. Degrave, C. Rougeot, A. Cosson, N. Chamond, A. Cordeiro-da-Silva, M. Arala-Chaves, A. Coutinho, and P. Minoprio (2000) A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase. Nature Medicine 6: 890–897.CrossRefPubMedGoogle Scholar
  31. 31.
    de Oliveira, C. A. F., B. J. Grant, M. Zhou, and J. A. McCammon (2011) Large-scale conformational changes of Trypanosoma cruzi proline racemase predicted by accelerated molecular dynamics simulation. PLoS Computational Biology 7: e1002178.CrossRefGoogle Scholar
  32. 32.
    Goytia, M., N. Chamond, A. Cosson, N. Coatnoan, D. Hermant, A. Berneman, and P. Minoprio (2007) Molecular and structural discrimination of proline racemase and hydroxyproline-2-epimerase from nosocomial and bacterial pathogens. PLoS One 2: e885.CrossRefGoogle Scholar
  33. 33.
    Park, J. C., J. C. Joo, E. S. An, B. K. Song, Y. H. Kim, and Y. J. Yoo (2011) A combined approach of experiments and computational docking simulation to the Coprinus cinereus peroxidasecatalyzed oxidative polymerization of alkyl phenols. Bioresource Technology 102: 4901–4904.CrossRefPubMedGoogle Scholar
  34. 34.
    Cho, S. J., J. A. Kim, and S. B. Lee (2015) Identification and characterization of 3,6-anhydro-L-galactonate cycloisomerase belonging to theenolase superfamily. Biotechnol. Bioproc. E 20: 462–472.CrossRefGoogle Scholar
  35. 35.
    Mortazavi, S. S., D. Chavez-Flores, and J. M. Salvador (2016) Isomerase activity of Candida rugosa lipase in the optimized conversion of racemic ibuprofen to (S)-ibuprofen. Biotechnol. Bioproc. E 21: 634–640.CrossRefGoogle Scholar

Copyright information

© The Korean Society for Biotechnology and Bioengineering and Springer 2019

Authors and Affiliations

  • Sungmin Byun
    • 1
  • Hyun June Park
    • 2
  • Jeong Chan Joo
    • 3
  • Yong Hwan Kim
    • 1
    Email author
  1. 1.School of Energy and Chemical EngineeringUNISTUlsanKorea
  2. 2.Bio-Max InstituteSeoul National UniversitySeoulKorea
  3. 3.Korea Research Institute of Chemical TechnologyDaejeonKorea

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