Applied Microbiology and Biotechnology

, Volume 94, Issue 5, pp 1221–1231 | Cite as

Stereoselective hydrolysis of aryl-substituted dihydropyrimidines by hydantoinases

Biotechnologically relevant enzymes and proteins


In this study, we investigated the possibility of using a modified hydantoinase process for the production of optically pure β-amino acids. Two aryl-substituted dihydropyrimidines d,l-6-phenyl-5,6-dihydrouracil (PheDU) and para-chloro-d,l-6-phenyl-5,6-dihydrouracil (pClPheDU) were synthesized. Hydrolysis of these novel substrates to the corresponding N-carbamoyl-β-amino acids by three recombinant d-hydantoinases and several bacterial strains was tested. All applied recombinant d-hydantoinases and eight bacterial isolates catalyzed the conversion of PheDU to N-carbamoyl-β-phenylalanine (NCβPhe). Some of these biocatalysts showed an enantioselectivity for either the d- or the l-PheDU enantiomer. The second dihydropyrimidinase substrate pClPheDU was hydrolyzed by all three recombinant d-hydantoinases and six of the wild-type strains. To our knowledge, this is the first dihydropyrimidinase activity reported with this aryl-substituted dihydropyrimidine. For selected biocatalysts, hydantoinase activity towards aryl-substituted hydantoins was demonstrated as well. However, none of the bacterial strains tested so far exhibited any carbamoylase activity towards NCβPhe.


Beta-amino acid Hydantoinase Dihydropyrimidinase Carbamoylase 



This work was financed by the Federal Ministry of Science and Education (BMBF), Germany. Furthermore, the authors like to thank Gerd Unkelbach from Fraunhofer Institute for Chemical Technology for cooperation in the synthesis of the above-mentioned substrates. We also thank Professor Don Cowan for providing the soil samples for the screening experiments.


  1. Andújar-Sánchez M, Las Heras-Vázquez FJ, Clemente-Jiménez JM, Martínez-Rodríguez S, Camara-Artigas A, Rodríguez-Vico F, Jara-Pérez V (2006) Enzymatic activity assay of d-hydantoinase by isothermal titration calorimetry. Determination of the thermodynamic activation parameters for the hydrolysis of several substrates. J Biochem Biophys Methods 67(1):57–66CrossRefGoogle Scholar
  2. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL (2006) GenBank. Nucleic Acids Res 34:D16–D20CrossRefGoogle Scholar
  3. Bertani G (1951) Studies on lysogenesis.1. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol 62(3):293–300Google Scholar
  4. Cai Y, Trodler P, Jiang S, Zhang W, Wu Y, Lu Y, Yang S, Jiang W (2009) Isolation and molecular characterization of a novel d-hydantoinase from Jannaschia sp. CCS1. FEBS J 276(13):3575–3588. doi: 10.1111/j.1742-4658.2009.07077.x CrossRefGoogle Scholar
  5. Cheng RP, Gellman SH, DeGrado WF (2001) β-Peptides: from structure to function. Chem Reviews 101(10):3219–3232. doi: 10.1021/cr000045i CrossRefGoogle Scholar
  6. Dakin HD, Dudley HW (1914) The resolution of inactive uramido-acids and hydantoins into active components, and their conversion into amino-acids. I. β-Phenyl-α-uramidopropionic acid, benzylhydantoin and phenylalanine. J Biol Chem 17(1):29–36Google Scholar
  7. Dürr R (2007) Screening and description of novel hydantoinases from distinct environmental sources. Doctoral thesis, Universität Karlsruhe (TH), Karlsruhe, ISBN 9783866441736Google Scholar
  8. Dürr R, Vielhauer O, Burton SG, Cowan DA, Punal A, Brandao PFB, Bull AT, Syldatk C (2006) Distribution of hydantoinase activity in bacterial isolates from geographically distinct environmental sources. J Mol Catal B Enzym 39(1–4):160–165CrossRefGoogle Scholar
  9. Dürr R, Neumann A, Vielhauer O, Altenbuchner J, Burton SG, Cowan DA, Syldatk C (2008) Genes responsible for hydantoin degradation of a halophilic Ochrobactrum sp. G21 and Delftia sp. 124—new insight into relation of d-hydantoinases and dihydropyrimidinases. J Mol Catal B Enzym 52–3:2–12CrossRefGoogle Scholar
  10. Fischer E, Roeder G (1901) Synthese des Uracils, Thymins und Phenyluracils. Ber Dtsch Chem Ges 34(3):3751–3763CrossRefGoogle Scholar
  11. Fleming PE, Mocek U, Floss HG (1993) Biosynthesis of taxoids. Mode of formation of the taxol side chain. J Am Chem Soc 115(2):805–807. doi: 10.1021/ja00055a072 CrossRefGoogle Scholar
  12. Frackenpohl J, Arvidsson PI, Schreiber JV, Seebach D (2001) The outstanding biological stability of β- and γ-peptides toward proteolytic enzymes: an in vitro investigation with fifteen peptidases. ChemBioChem 2(6):445–455CrossRefGoogle Scholar
  13. Gokhale DV, Bastawde KB, Patil SG, Kalkote UR, Joshi RR, Joshi RA, Ravindranathan T, Gaikwad BG, Jogdand VV, Nene S (1996) Chemoenzymatic synthesis of d-phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells. Enzyme Microb Technol 18(5):353–357CrossRefGoogle Scholar
  14. Kao C-H, Lo H-H, Hsu S-K, Hsu W-H (2008) A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and l-N-carbamoylase activities as biocatalyst for the production of l-homophenylalanine. J Biotechnol 134(3–4):231CrossRefGoogle Scholar
  15. Lane D (1991) 16S/23S rRNA Sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, ChichesterGoogle Scholar
  16. Liljeblad A, Kanerva LT (2006) Biocatalysis as a profound tool in the preparation of highly enantiopure β-amino acids. Tetrahedron 62(25):5831–5854CrossRefGoogle Scholar
  17. Liu M, Sibi MP (2002) Recent advances in the stereoselective synthesis of β-amino acids. Tetrahedron 58(40):7991–8035CrossRefGoogle Scholar
  18. Louwrier A, Knowles CJ (1996) The purification and characterization of a novel d-specific carbamoylase enzyme from an Agrobacterium sp. Enzyme Microb Technol 19(8):562–571CrossRefGoogle Scholar
  19. Martínez-Gómez AI, Martínez-Rodríguez S, Clemente-Jiménez JM, Pozo-Dengra J, Rodríguez-Vico F, Las Heras-Vázquez FJ (2007) Recombinant polycistronic structure of hydantoinase process genes in Escherichia coli for the production of optically pure d-amino acids. Appl Environ Microbiol 73(5):1525–1531. doi: 10.1128/aem.02365-06 CrossRefGoogle Scholar
  20. Martínez-Gómez AI, Martínez-Rodríguez S, Pozo-Dengra J, Tessaro D, Servi S, Clemente-Jiménez JM, Rodríguez-Vico F, Las Heras-Vázquez FJ (2009) Potential application of N-carbamoyl-β-alanine amidohydrolase from Agrobacterium tumefaciens C58 for β-amino acid production. Appl Environ Microbiol 75(2):514–520. doi: 10.1128/aem.01128-08 CrossRefGoogle Scholar
  21. Martínez-Rodríguez S, Martínez-Gómez AI, Clemente-Jiménez JM, Rodríguez-Vico F, Garcia-Ruiz JM, Las Heras-Vázquez FJ, Gavira JA (2010) Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: new features in an amidohydrolase family member. J Struct Biol 169(2):200–208. doi: 10.1016/j.jsb.2009.10.013 CrossRefGoogle Scholar
  22. May O, Siemann M, Pietzsch M, Kiess M, Mattes R, Syldatk C (1998) Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases. J Biotechnol 61(1):1CrossRefGoogle Scholar
  23. Ogawa J, Shimizu S (1994) β-Ureidopropionase with N-carbamoyl-α-l-amino acid amidohydrolase activity from an aerobic bacterium, Pseudomonas putida IFO 12996. Eur J Biochem 223(2):625–630CrossRefGoogle Scholar
  24. Rehdorf J, Mihovilovic MD, Bornscheuer UT (2010) Exploiting the regioselectivity of Baeyer–Villiger monooxygenases for the formation of β-amino acids and β-amino alcohols. Angewandte Chemie International Edition 49(26):4506–4508. doi: 10.1002/anie.201000511 CrossRefGoogle Scholar
  25. Schnackerz KD, Dobritzsch D (2008) Amidohydrolases of the reductive pyrimidine catabolic pathway: purification, characterization, structure, reaction mechanisms and enzyme deficiency. Biochim Biophys Acta, Proteins Proteomics 1784(3):431–444CrossRefGoogle Scholar
  26. Scholl S, Koch A, Henning D, Kempter G, Kleinpeter E (1999) The influence of structure and lipophilicity of hydantoin derivatives on anticonvulsant activity. Struct Chem 10(5):355–366. doi: 10.1023/a:1022091411018 CrossRefGoogle Scholar
  27. Seebach D, Gardiner J (2008) β-Peptidic peptidomimetics. Acc Chem Res 41(10):1366–1375. doi: 10.1021/ar700263g CrossRefGoogle Scholar
  28. Seebach D, Matthews JL (1997) β-Peptides: a surprise at every turn. Chem Commun (Cambridge, UK) 21:2015–2022CrossRefGoogle Scholar
  29. Siemann M, Alvarado-Marin A, Pietzsch M, Syldatk C (1999) A d-specific hydantoin amidohydrolase: properties of the metalloenzyme purified from Arthrobacter crystallopoietes. J Mol Catal B: Enzym 6(3):387CrossRefGoogle Scholar
  30. Stark GR, Smyth DG (1963) Use of cyanate for determination of NH2-terminal residues in proteins. J Biol Chem 238(1):214–226Google Scholar
  31. Steer DL, Lew RA, Perlmutter P, Smith AI, Aguilar M-I (2002) β2-Amino acids: versatile peptidomimetics. Curr Med Chem 9:811CrossRefGoogle Scholar
  32. Suzuki S, Henderson PJF (2006) The hydantoin transport protein from Microbacterium liquefaciens. J Bacteriol 188(9):3329–3336. doi: 10.1128/Jb.188.9.3329-3336.2006 CrossRefGoogle Scholar
  33. Suzuki T, Igarashi K, Hase K, Tuzimura K (1973) Optical-rotatory dispersion and circular-dichroism of amino-acid hydantoins. Agric Biol Chem 37(2):411–416CrossRefGoogle Scholar
  34. Syldatk C, Mackowiak V, Hoke H, Gross C, Dombach G, Wagner F (1990) Cell growth and enzyme synthesis of a mutant of Arthrobacter sp. (DSM 3747) used for the production of l-amino acids from dl-5-monosubstituted hydantoins. J Biotechnol 14(3–4):345CrossRefGoogle Scholar
  35. Tasnádi G, Forró E, Fülöp F (2008) An efficient new enzymatic method for the preparation of β-aryl-β-amino acid enantiomers. Tetrahedron: Asymmetry 19(17):2072–2077CrossRefGoogle Scholar
  36. Vogels GD, van der Drift C (1976) Degradation of purines and pyrimidines by microorganisms. Bacteriological Reviews 40(2):403–468Google Scholar
  37. Vuano BM, Pieroni OI, Cabaleiro MC (2000) Thermal reaction of cinnamic acid and of β-styrylphosphonic acid with urea. J Chem Res 7:318–320CrossRefGoogle Scholar
  38. Watabe K, Ishikawa T, Mukohara Y, Nakamura H (1992) Cloning and sequencing of the genes involved in the conversion of 5-substituted hydantoins to the corresponding l-amino acids from the native plasmid of Pseudomonas sp. strain NS671. J Bacteriol 174(3):962–969Google Scholar
  39. Weiner B, Szymanski W, Janssen DB, Minnaard AJ, Feringa BL (2010) Recent advances in the catalytic asymmetric synthesis of β-amino acids. Chem Soc Rev 39(5):1656–1691CrossRefGoogle Scholar
  40. Werner M, Las Heras-Vazques FJ, Fritz C, Vielhauer O, Siemann-Herzberg M, Altenbuchner J, Syldatk C (2004) Cloning of d-specific hydantoin utilization genes from Arthrobacter crystallopoietes. Eng Life Sci 4(6):563–572CrossRefGoogle Scholar
  41. Wu B, Szymanski W, Wietzes P, de Wildeman S, Poelarends GJ, Feringa BL, Janssen DB (2009) Enzymatic synthesis of enantiopure α- and β-amino acids by phenylalanine aminomutase-catalysed amination of cinnamic acid derivatives. ChemBioChem 10(2):338–344. doi: 10.1002/cbic.200800568 CrossRefGoogle Scholar
  42. Yokozeki K, Kubota K (1987) Enzymatic production of d-amino acids from 5-substituted hydantoins: 3. Mechanism of asymmetric production of d-amino acids from the corresponding hydantoins by Pseudomonas sp. Agric Biol Chem 51(3):721–728CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  1. 1.Karlsruhe Institute of Technology (KIT)Institute of Process Engineering in Life Sciences, Section II: Technical BiologyKarlsruheGermany

Personalised recommendations