Advertisement

Applied Microbiology and Biotechnology

, Volume 100, Issue 7, pp 3101–3111 | Cite as

Identification of novel thermostable taurine–pyruvate transaminase from Geobacillus thermodenitrificans for chiral amine synthesis

  • Yujie Chen
  • Dong YiEmail author
  • Shuiqin Jiang
  • Dongzhi WeiEmail author
Biotechnologically relevant enzymes and proteins

Abstract

ω-Transaminases (ω-TAs) are one of the most popular candidate enzymes in the biosynthesis of chiral amines. Determination of yet unidentified ω-TAs is important to broaden their potential for synthetic application. Taurine–pyruvate TA (TPTA, EC 2.6.1.77) is an ω-TA belonging to class III of TAs. In this study, we cloned a novel thermostable TPTA from Geobacillus thermodenitrificans (TPTAgth) and overexpressed it in Escherichia coli. The enzyme showed the highest activity at pH 9.0 and 65 °C, with remarkable thermostability and tolerance toward organic solvents. Its K M and v max values for taurine were 5.3 mM and 0.28 μmol s−1 mg−1, respectively. Determination of substrate tolerance indicated its broad donor and acceptor ranges for unnatural substrates. Notably, the enzyme showed relatively good activity toward ketoses, suggesting its potential for catalyzing the asymmetric synthesis of chiral amino alcohols. The active site of TPTAgth was identified by performing protein sequence alignment, three-dimensional structure simulation, and coenzyme pyridoxamine phosphate docking. The protein sequence and structure of TPTAgth were similar to those of TAs belonging to the 3N5M subfamily. Its active site was found to be its special large pocket and substrate tunnel. In addition, TPTAgth showed a unique mechanism of sulfonate/α-carboxylate recognition contributed by Arg163 and Gln160. We also determined the protein sequence fingerprint of TPTAs in the 3N5M subfamily, which involved Arg163 and Gln160 and seven additional residues from 413 to 419 and lacked Phe/Tyr22, Phe85, and Arg409.

Keywords

Taurine–pyruvate transaminase ω-Transaminase Biocatalysis Thermostable transaminase Chiral amine 

Notes

Acknowledgments

This work was funded by the National Natural Science Funds of China (Grant No. 21406069).

Conflict of interest

Mr. Yujie Chen declares that he has no conflict of interest.

Dr. Dong Yi declares that he has no conflict of interest.

Ms. Shuiqin Jiang declares that she has no conflict of interest.

Prof. Dr. Dongzhi Wei declares that he has no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2015_7129_MOESM1_ESM.pdf (100 kb)
Fig. S1 Nucleotide sequence of TPTAgth. The nucleotide sequence of TPTAgth has been deposited in the GenBank database under accession number KT719298. (PDF 99 kb)
253_2015_7129_MOESM2_ESM.pdf (152 kb)
Fig. S2 SDS-PAGE analysis of purified recombinant TPTAgth. (PDF 151 kb)
253_2015_7129_MOESM3_ESM.pdf (144 kb)
Fig. S3 Protein sequence alignment of TPTAgth. ω-TApde: ω-TA from Paracoccus denitrificans (UniProt ID: A1B956), ω-TAvfl: ω-TA from Vibrio fluvialis (UniProt ID: F2XBU9), ω-TAcvi: ω-TA from Chromobacterium violaceum (UniProt ID: Q7NWG4), ω-TAppu: ω-TA from Pseudomonas putida (UniProt ID: P28269), 3N5M: PDB item 3N5M, TPTAban: TPTA from Bacillus anthracis (UniProt ID: Q81SL2), TPTAbsu: TPTA from Bacillus subtilis (UniProt ID: P33189), TPTAbwa: TPTA from Bilophila wadsworthia (UniProt ID: Q9APM5), TPTApde: TPTA from Paracoccus denitrificans (UniProt ID: A1B9Z3), TPTArpo: TPTA from Ruegeria pomeroyi (UniProt ID: Q5LVM7). The sequence alignment was achieved by NTI Vector software 11.5 (ThermoFischer, USA). The identical residues are marked as *. The key residues are highlighted in bold. Arg163, Gln160 and the additional loop between α15 and β13 are highlighted in grey. (PDF 143 kb)

References

  1. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. doi: 10.1093/bioinformatics/bti770 CrossRefPubMedGoogle Scholar
  2. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Cassarino TG, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252–W258. doi: 10.1093/nar/gku340 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Breuer M, Ditrich K, Habicher T, Hauer B, Kesseler M, Sturmer R, Zelinski T (2004) Industrial methods for the production of optically active intermediates. Angew Chem Int Ed Engl 43:788–824. doi: 10.1002/anie.200300599 CrossRefPubMedGoogle Scholar
  4. Bruins ME, Janssen AE, Boom RM (2001) Thermozymes and their applications: a review of recent literature and patents. Appl Biochem Biotechnol 90:155–186. doi: 10.1385/ABAB:90:2:155 CrossRefPubMedGoogle Scholar
  5. Chien C, Leadbetter ER, Godchaux W (1997) Taurine-sulfur assimilation and taurine-pyruvate aminotransferase activity in anaerobic bacteria. Appl Environ Microbiol 63:3021–3024, Retrieved from http://aem.asm.org/content/63/8/3021.abstract?sid=4806b8f9-eb22-433d-ba1a-d40b2e2f161c PubMedPubMedCentralGoogle Scholar
  6. Cihan AC, Ozcan B, Tekin N, Cokmus C (2011) Geobacillus thermodenitrificans subsp. calidus, subsp. nov., a thermophilic and α-glucosidase producing bacterium isolated from Kizilcahamam, Turkey. J Gen Appl Microbiol 57:83–92. doi: 10.2323/jgam.57.83 CrossRefPubMedGoogle Scholar
  7. Cook AM, Denger K (2002) Dissimilation of the C2 sulfonates. Arch Microbiol 179:1–6. doi: 10.1007/s00203-002-0497-0 CrossRefPubMedGoogle Scholar
  8. Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P (2006) Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem 357:289–298. doi: 10.1016/j.ab.2006.07.027 CrossRefPubMedGoogle Scholar
  9. Felux AK, Denger K, Weiss M, Cook AM, Schleheck D (2013) Paracoccus denitrificans PD1222 utilizes hypotaurine via transamination followed by spontaneous desulfination to yield acetaldehyde, and finally, acetate for growth. J Bacteriol 195:2921–2930. doi: 10.1128/JB.00307-13 CrossRefPubMedPubMedCentralGoogle Scholar
  10. Gorzynska AK, Denger K, Cook AM, Smits TH (2006) Inducible transcription of genes involved in taurine uptake and dissimilation by Silicibacter pomeroyi DSS-3T. Arch Microbiol 185:402–406. doi: 10.1007/s00203-006-0106-8 CrossRefPubMedGoogle Scholar
  11. Gotor-Fernandez V, Gotor V (2009) Biocatalytic routes to chiral amines and amino acids. Curr Opin Drug Discov Devel 12:784–797, Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19894190 PubMedGoogle Scholar
  12. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30(Suppl 1):S162–173. doi: 10.1002/elps.200900140 CrossRefPubMedGoogle Scholar
  13. Hohn M, Bornscheuer UT (2009) Biocatalytic routes to optically active amines. Chemcatchem 1:42–51. doi: 10.1002/cctc.200900110 CrossRefGoogle Scholar
  14. Hohne M, Schatzle S, Jochens H, Robins K, Bornscheuer UT (2010) Rational assignment of key motifs for function guides in silico enzyme identification. Nat Chem Biol 6:807–813. doi: 10.1038/nchembio.447 CrossRefPubMedGoogle Scholar
  15. Huxtable RJ (1992) Physiological actions of taurine. Physiol Rev 72:101–163, Retrieved from http://physrev.physiology.org/content/72/1/101 PubMedGoogle Scholar
  16. Hwang BY, Ko SH, Park HY, Seo JH, Lee BS, Kim BG (2008) Identification of ω-aminotransferase from Caulobacter crescentus and site-directed mutagenesis to broaden substrate specificity. J Microbiol Biotechnol 18:48–54, Retrieved from http://www.jmb.or.kr/journal/viewJournal.html?year=2008&vol=18&num=1&page=48 PubMedGoogle Scholar
  17. Ingram CU, Bommer M, Smith MEB, Dalby PA, Ward JM, Hailes HC, Lye GJ (2007) One-pot synthesis of amino-alcohols using a de-novo transketolase and β-alanine: pyruvate transaminase pathway in Escherichia coli. Biotechnol Bioeng 96:559–569. doi: 10.1002/bit.21125 CrossRefPubMedGoogle Scholar
  18. Iwasaki A, Yamada Y, Ikenaka Y, Hasegawa J (2003) Microbial synthesis of (R)- and (S)-3,4-dimethoxyamphetamines through stereoselective transamination. Biotechnol Lett 25:1843–1846. doi: 10.1023/A:1026229610628 CrossRefPubMedGoogle Scholar
  19. Iwasaki A, Yamada Y, Kizaki N, Ikenaka Y, Hasegawa J (2006) Microbial synthesis of chiral amines by (R)-specific transamination with Arthrobacter sp. KNK168. Appl Microbiol Biotechnol 69:499–505. doi: 10.1007/s00253-005-0002-1 CrossRefPubMedGoogle Scholar
  20. Jansonius JN (1998) Structure, evolution and action of vitamin B6-dependent enzymes. Curr Opin Struct Biol 8:759–769. doi: 10.1016/S0959-440X(98)80096-1 CrossRefPubMedGoogle Scholar
  21. Jiang JJ, Chen X, Feng JH, Wu QQ, Zhu DM (2014) Substrate profile of an ω-transaminase from Burkholderia vietnamiensis and its potential for the production of optically pure amines and unnatural amino acids. J Mol Catal B Enzym 100:32–39. doi: 10.1016/j.molcatb.2013.11.013 CrossRefGoogle Scholar
  22. Kaulmann U, Smithies K, Smith MEB, HaileS HC, Ward JM (2007) Substrate spectrum of ω-transaminase from Chromobacterium violaceum DSM30191 and its potential for biocatalysis. Enzyme Microb Tech 41:628–637. doi: 10.1016/j.enzmictec.2007.05.011 CrossRefGoogle Scholar
  23. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Res 37:D387–392. doi: 10.1093/nar/gkn750 CrossRefPubMedPubMedCentralGoogle Scholar
  24. Koszelewski D, Tauber K, Faber K, Kroutil W (2010) ω-Transaminases for the synthesis of non-racemic α-chiral primary amines. Trends Biotechnol 28:324–332. doi: 10.1016/j.tibtech.2010.03.003 CrossRefPubMedGoogle Scholar
  25. Laue H, Cook AM (2000) Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobe Bilophila wadsworthia. Eur J Biochem 267:6841–6848. doi: 10.1046/j.1432-1033.2000.01782.x CrossRefPubMedGoogle Scholar
  26. Malik MS, Park ES, Shin JS (2012) Features and technical applications of ω-transaminases. Appl Microbiol Biotechnol 94:1163–1171. doi: 10.1007/s00253-012-4103-3 CrossRefPubMedGoogle Scholar
  27. Mikosch CA, Denger K, Schafer EM, Cook AM (1999) Anaerobic oxidations of cysteate: degradation via L-cysteate:2-oxoglutarate aminotransferase in Paracoccus pantotrophus. Microbiology 145(Pt 5):1153–1160. doi: 10.1099/13500872-145-5-1153 CrossRefPubMedGoogle Scholar
  28. Mutti FG, Fuchs CS, Pressnitz D, Sattler JH, Kroutil W (2011) Stereoselectivity of four (R)-Selective transaminases for the asymmetric amination of ketones. Adv Synth Catal 353:3227–3233. doi: 10.1002/adsc.201100558 CrossRefGoogle Scholar
  29. Nazina TN, Tourova TP, Poltaraus AB, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko AM, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV (2001) Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51:433–446. doi: 10.1099/00207713-51-2-433 CrossRefPubMedGoogle Scholar
  30. Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221. doi: 10.1038/nprot.2007.321 CrossRefPubMedGoogle Scholar
  31. Novak RT, Gritzer RF, Leadbetter ER, Godchaux W (2004) Phototrophic utilization of taurine by the purple nonsulfur bacteria Rhodopseudomonas palustris and Rhodobacter sphaeroides. Microbiology 150:1881–1891. doi: 10.1099/mic.0.27023-0 CrossRefPubMedGoogle Scholar
  32. Park E, Kim M, Shin JS (2010) One-pot conversion of L-threonine into L-homoalanine: biocatalytic production of an unnatural amino acid from a natural one. Adv Synth Catal 352:3391–3398. doi: 10.1002/adsc.201000601 CrossRefGoogle Scholar
  33. Park ES, Kim M, Shin JS (2012) Molecular determinants for substrate selectivity of ω-transaminases. Appl Microbiol Biotechnol 93:2425–2435. doi: 10.1007/s00253-011-3584-9 CrossRefPubMedGoogle Scholar
  34. Percudani R, Peracchi A (2009) 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. doi: 10.1186/1471-2105-10-273 CrossRefPubMedPubMedCentralGoogle Scholar
  35. Rausch C, Lerchner A, Schiefner A, Skerra A (2013) Crystal structure of the ω-aminotransferase from Paracoccus denitrificans and its phylogenetic relationship with other class III aminotransferases that have biotechnological potential. Proteins 81:774–787. doi: 10.1002/prot.24233 CrossRefPubMedGoogle Scholar
  36. Savile CK, Janey JM, Mundorff EC, Moore JC, Tam S, Jarvis WR, Colbeck JC, Krebber A, Fleitz FJ, Brands J, Devine PN, Huisman GW, Hughes GJ (2010) Biocatalytic asymmetric synthesis of chiral amines from ketones applied to sitagliptin manufacture. Science 329:305–309. doi: 10.1126/science.1188934 CrossRefPubMedGoogle Scholar
  37. Schatzle S, Hohne M, Redestad E, Robins K, Bornscheuer UT (2009) Rapid and sensitive kinetic assay for characterization of ω-transaminases. Anal Chem 81:8244–8248. doi: 10.1021/ac901640q CrossRefPubMedGoogle Scholar
  38. Schatzle S, Steffen-Munsberg F, Thontowi A, Hohne M, Robins K, Bornscheuer UT (2011) Enzymatic asymmetric synthesis of enantiomerically pure aliphatic, aromatic and arylaliphatic amines with (R)-selective amine transaminases. Adv Synth Catal 353:2439–2445. doi: 10.1002/adsc.201100435 CrossRefGoogle Scholar
  39. Scheidt T, Land H, Anderson M, Chen YJ, Berglund P, Yi D, Fessner WD (2015) Fluorescence-based kinetic assay for high-throughput discovery and engineering of stereoselective ω-transaminases. Adv Synth Catal 357:1721–1731. doi: 10.1002/adsc.201500215 CrossRefGoogle Scholar
  40. Shin JS, Kim BG (1997) Kinetic resolution of α-methylbenzylamine with ω-transaminase screened from soil microorganisms: application of a biphasic system to overcome product inhibition. Biotechnol Bioeng 55:348–358. doi: 10.1002/(SICI)1097-0290(19970720)55:2<348::AID-BIT12>3.0.CO;2-D CrossRefPubMedGoogle Scholar
  41. Shin JS, Kim BG (2002) Exploring the active site of amine: pyruvate aminotransferase on the basis of the substrate structure-reactivity relationship: how the enzyme controls substrate specificity and stereo selectivity. J Org Chem 67:2848–2853. doi: 10.1021/jo016115i CrossRefPubMedGoogle Scholar
  42. Shin JS, Yun H, Jang JW, Park I, Kim BG (2003) Purification, characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17. Appl Microbiol Biotechnol 61:463–471. doi: 10.1007/s00253-003-1250-6 CrossRefPubMedGoogle Scholar
  43. Steffen-Munsberg F, Vickers C, Kohls H, Land H, Mallin H, Nobili A, Skalden L, van den Bergh T, Joosten HJ, Berglund P, Höhne M, Bornscheuer UT (2015) Bioinformatic analysis of a PLP-dependent enzyme superfamily suitable for biocatalytic applications. Biotechnol Adv 33:566–604. doi: 10.1016/j.biotechadv.2014.12.012 CrossRefPubMedGoogle Scholar
  44. Svedendahl M, Branneby C, Lindberg L, Berglund P (2010) Reversed enantiopreference of an ω-transaminase by a single-point mutation. Chemcatchem 2:976–980. doi: 10.1002/cctc.201000107 CrossRefGoogle Scholar
  45. von Rekowski KS, Denger K, Cook AM (2005) Isethionate as a product from taurine during nitrogen-limited growth of Klebsiella oxytoca TauN1. Arch Microbiol 183:325–330. doi: 10.1007/s00203-005-0776-7 CrossRefGoogle Scholar
  46. Ward J, Wohlgemuth R (2010) High-yield biocatalytic amination reactions in organic synthesis. Curr Org Chem 14:1914–1927. doi: 10.2174/138527210792927546 CrossRefGoogle Scholar
  47. Watanabe N, Sakabe K, Sakabe N, Higashi T, Sasaki K, Aibara S, Morita Y, Yonaha K, Toyama S, Fukutani H (1989) Crystal structure analysis of ω-amino acid:pyruvate aminotransferase with a newly developed Weissenberg camera and an imaging plate using synchrotron radiation. J Biochem 105:1–3, Retrieved from https://www.jstage.jst.go.jp/article/biochemistry1922/105/1/105_1_1/_article PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.State Key Laboratory of Bioreactor EngineeringEast China University of Science and TechnologyShanghaiPeople’s Republic of China

Personalised recommendations