Applied Microbiology and Biotechnology

, Volume 100, Issue 10, pp 4511–4521 | Cite as

Bacillus anthracis ω-amino acid:pyruvate transaminase employs a different mechanism for dual substrate recognition than other amine transaminases

  • Fabian Steffen-Munsberg
  • Philipp Matzel
  • Miriam A. Sowa
  • Per Berglund
  • Uwe T. Bornscheuer
  • Matthias Höhne
Applied genetics and molecular biotechnology

Abstract

Understanding the metabolic potential of organisms or a bacterial community based on their (meta) genome requires the reliable prediction of an enzyme’s function from its amino acid sequence. Besides a remarkable development in prediction algorithms, the substrate scope of sequences with low identity to well-characterized enzymes remains often very elusive. From a recently conducted structure function analysis study of PLP-dependent enzymes, we identified a putative transaminase from Bacillus anthracis (Ban-TA) with the crystal structure 3N5M (deposited in the protein data bank in 2011, but not yet published). The active site residues of Ban-TA differ from those in related (class III) transaminases, which thereby have prevented function predictions. By investigating 50 substrate combinations its amine and ω-amino acid:pyruvate transaminase activity was revealed. Even though Ban-TA showed a relatively narrow amine substrate scope within the tested substrates, it accepts 2-propylamine, which is a prerequisite for industrial asymmetric amine synthesis. Structural information implied that the so-called dual substrate recognition of chemically different substrates (i.e. amines and amino acids) differs from that in formerly known enzymes. It lacks the normally conserved ‘flipping’ arginine, which enables dual substrate recognition by its side chain flexibility in other ω-amino acid:pyruvate transaminases. Molecular dynamics studies suggested that another arginine (R162) binds ω-amino acids in Ban-TA, but no side chain movements are required for amine and amino acid binding. These results, supported by mutagenesis studies, provide functional insights for the B. anthracis enzyme, enable function predictions of related proteins, and broadened the knowledge regarding ω-amino acid and amine converting transaminases.

Keywords

Enzyme catalysis Transamination Functional analysis Structure activity relationship 

Supplementary material

253_2015_7275_MOESM1_ESM.pdf (10.8 mb)
ESM 1(PDF 10.7 mb)

References

  1. Bommer M, Ward JM (2013) A 1-step microplate method for assessing the substrate range of L-α-amino acid aminotransferase. Enzyme Microb Tech 52:218–225. doi:10.1016/j.enzmictec.2013.02.007 CrossRefGoogle Scholar
  2. Cassimjee KE, Manta B, Himo F (2015) A quantum chemical study of the ω-transaminase reaction mechanism. Org Biomol Chem 13:8453–8464. doi:10.1039/c5ob00690b CrossRefPubMedGoogle Scholar
  3. Crismaru CG, Wybenga GG, Szymanski W, Wijma HJ, Wu B, Bartsch S, de Wildeman S, Poelarends GJ, Feringa BL, Dijkstra BW, Janssen DB (2013) Biochemical properties and crystal structure of a β-phenylalanine aminotransferase from Variovorax paradoxus. Appl Environ Microbiol 79:185–195. doi:10.1128/aem.02525-12 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Davies MT (1959) A universal buffer solution for use in ultra-violet spectrophotometry. Analyst 84:248–251. doi:10.1039/an9598400248 CrossRefGoogle Scholar
  5. Dey S, Lane JM, Lee RE, Rubin EJ, Sacchettini JC (2010) Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase. Biochemistry 49:6746–6760. doi:10.1021/bi902097j CrossRefPubMedPubMedCentralGoogle Scholar
  6. Duan Y, Wu C, Chowdhury S, Lee MC, Xiong G, Zhang W, Yang R, Cieplak P, Luo R, Lee T, Caldwell J, Wang J, Kollman P (2003) A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J Comput Chem 24:1999–2012. doi:10.1002/jcc.10349 CrossRefPubMedGoogle Scholar
  7. Engelmark Cassimjee K, Branneby C, Abedi V, Wells A, Berglund P (2010) Transaminations with isopropyl amine: equilibrium displacement with yeast alcohol dehydrogenase coupled to in situ cofactor regeneration. Chem Comm 46:5569–5571. doi:10.1039/c0cc00050g CrossRefGoogle Scholar
  8. Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG (1995) A smooth particle mesh Ewald method. J Chem Phys 103:8577–8593. doi:10.1063/1.470117 CrossRefGoogle Scholar
  9. Gand M, Müller H, Wardenga R, Höhne M (2014) Characterization of three novel enzymes with imine reductase activity. J Mol Catal B Enzym 110:126–132. doi:10.1016/j.molcatb.2014.09.017 CrossRefGoogle Scholar
  10. Höhne M, Bornscheuer UT (2012) Application of transaminases in organic synthesis. In: May O, Gröger H, Drauz W (eds) Enzymes in organic synthesis. Wiley-VCH, Weinheim, pp. 779–820Google Scholar
  11. Höhne M, Schätzle 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
  12. Ivanova N, Sorokin A, Anderson I, Galleron N, Candelon B, Kapatral V, Bhattacharyya A, Reznik G, Mikhailova N, Lapidus A, Chu L, Mazur M, Goltsman E, Larsen N, D’Souza M, Walunas T, Grechkin Y, Pusch G, Haselkorn R, Fonstein M, Dusko Ehrlich S, Overbeek R, Kyrpides N (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423:87–91. doi:10.1038/nature01582 CrossRefPubMedGoogle Scholar
  13. Kohls H, Steffen-Munsberg F, Höhne M (2014) Recent achievements in developing the biocatalytic toolbox for chiral amine synthesis. Curr Opin Chem Biol 19:180–192. doi:10.1016/j.cbpa.2014.02.021 CrossRefPubMedGoogle Scholar
  14. 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
  15. Midelfort KS, Kumar R, Han S, Karmilowicz MJ, McConnell K, Gehlhaar DK, Mistry A, Chang JS, Anderson M, Villalobos A, Minshull J, Govindarajan S, Wong JW (2013) Redesigning and characterizing the substrate specificity and activity of Vibrio fluvialis aminotransferase for the synthesis of imagabalin. Protein Eng Des Sel 26:25–33. doi:10.1093/protein/gzs065 CrossRefPubMedGoogle Scholar
  16. Nakano Y, Tokunaga H, Kitaoka S (1977) Two ω-amino acid transaminases from Bacillus cereus. J Biochem 81:1375–1381PubMedGoogle Scholar
  17. Økstad O, Kolstø A-B (2011) Genomics of Bacillus species. In: Wiedmann M, Zhang W (eds) Genomics of foodborne bacterial pathogens. Food Microbiology and Food Safety. Springer, New York, pp. 29–53. doi:10.1007/978-1-4419-7686-4_2 CrossRefGoogle Scholar
  18. Ono H, Sawada K, Khunajakr N, Tao T, Yamamoto M, Hiramoto M, Shinmyo A, Takano M, Murooka Y (1999) Characterization of biosynthetic enzymes for ectoine as a compatible solute in a moderately halophilic eubacterium, Halomonas elongata. J Bacteriol 181:91–99PubMedPubMedCentralGoogle Scholar
  19. Park E-S, Shin J-S (2013) ω-transaminase from Ochrobactrum anthropi is devoid of substrate and product inhibitions. Appl Environ Microbiol 79:4141–4144. doi:10.1128/aem.03811-12 CrossRefPubMedPubMedCentralGoogle Scholar
  20. Radivojac P, Clark WT, Oron TR, Schnoes AM, Wittkop T, Sokolov A, Graim K, Funk C, Verspoor K, Ben-Hur A, Pandey G, Yunes JM, Talwalkar AS, Repo S, Souza ML, Piovesan D, Casadio R, Wang Z, Cheng J, Fang H, Gough J, Koskinen P, Toronen P, Nokso-Koivisto J, Holm L, Cozzetto D, Buchan DWA, Bryson K, Jones DT, Limaye B, Inamdar H, Datta A, Manjari SK, Joshi R, Chitale M, Kihara D, Lisewski AM, Erdin S, Venner E, Lichtarge O, Rentzsch R, Yang H, Romero AE, Bhat P, Paccanaro A, Hamp T, Kaszner R, Seemayer S, Vicedo E, Schaefer C, Achten D, Auer F, Boehm A, Braun T, Hecht M, Heron M, Honigschmid P, Hopf TA, Kaufmann S, Kiening M, Krompass D, Landerer C, Mahlich Y, Roos M, Bjorne J, Salakoski T, Wong A, Shatkay H, Gatzmann F, Sommer I, Wass MN, Sternberg MJE, Skunca N, Supek F, Bosnjak M, Panov P, Dzeroski S, Smuc T, Kourmpetis YAI, van Dijk ADJ, Braak CJF, Zhou Y, Gong Q, Dong X, Tian W, Falda M, Fontana P, Lavezzo E, Di Camillo B, Toppo S, Lan L, Djuric N, Guo Y, Vucetic S, Bairoch A, Linial M, Babbitt PC, Brenner SE, Orengo C, Rost B, Mooney SD, Friedberg I (2013) A large-scale evaluation of computational protein function prediction. Nat Meth 10:221–227. doi:10.1038/nmeth.2340 CrossRefGoogle Scholar
  21. 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 Struct Funct Bioinf 81:774–787. doi:10.1002/prot.24233 CrossRefGoogle Scholar
  22. Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Tettelin H, Fouts DE, Eisen JA, Gill SR, Holtzapple EK, Okstad OA, Helgason E, Rilstone J, Wu M, Kolonay JF, Beanan MJ, Dodson RJ, Brinkac LM, Gwinn M, DeBoy RT, Madpu R, Daugherty SC, Durkin AS, Haft DH, Nelson WC, Peterson JD, Pop M, Khouri HM, Radune D, Benton JL, Mahamoud Y, Jiang L, Hance IR, Weidman JF, Berry KJ, Plaut RD, Wolf AM, Watkins KL, Nierman WC, Hazen A, Cline R, Redmond C, Thwaite JE, White O, Salzberg SL, Thomason B, Friedlander AM, Koehler TM, Hanna PC, Kolsto A-B, Fraser CM (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423:81–86. doi:10.1038/nature01586 CrossRefPubMedGoogle Scholar
  23. Sayer C, Bommer M, Isupov M, Ward J, Littlechild J (2012) Crystal structure and substrate specificity of the thermophilic serine: pyruvate aminotransferase from Sulfolobus solfataricus. Acta Crystallogr D Biol Crystallogr 68:763–772. doi:10.1107/s0907444912011274 CrossRefPubMedGoogle Scholar
  24. Schätzle S, Höhne 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
  25. Steffen-Munsberg F, Vickers C, Kohls H, Land H, Mallin H, Nobili A, Skalden L, van den Bergh T, Joosten HJ, Berglund P, Hohne 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
  26. Steffen-Munsberg F, Vickers C, Thontowi A, Schätzle S, Meinhardt T, Svedendahl Humble M, Land H, Berglund P, Bornscheuer UT, Höhne M (2013a) Revealing the structural basis of promiscuous amine transaminase activity. ChemCatChem 5:154–157. doi:10.1002/cctc.201200545 CrossRefGoogle Scholar
  27. Steffen-Munsberg F, Vickers C, Thontowi A, Schätzle S, Tumlirsch T, Svedendahl Humble M, Land H, Berglund P, Bornscheuer UT, Höhne M (2013b) Connecting unexplored protein crystal structures to enzymatic function. ChemCatChem 5:150–153. doi:10.1002/cctc.201200544 CrossRefGoogle Scholar
  28. Strecker HJ (1953) Glutamic dehydrogenase. Arch Biochem Biophys 46:128–140. doi:10.1016/0003-9861(53)90176-3 CrossRefPubMedGoogle Scholar
  29. Tamaki N, Kaneko M, Mizota C, Kikugawa M, Fujimoto S (1990) Purification, characterization and inhibition of D-3-aminoisobutyrate aminotransferase from the rat liver. Eur J Biochem 189:39–45. doi:10.1111/j.1432-1033.1990.tb15457.x CrossRefPubMedGoogle Scholar
  30. The PyMOL Molecular Graphics System, Version 1.6.0.0, Schrödinger, LCC, 2013Google Scholar
  31. Toney MD (2011) Controlling reaction specificity in pyridoxal phosphate enzymes. Biochim Biophys Acta, Proteins Proteomics 1814:1407–1418. doi:10.1016/j.bbapap.2011.05.019 CrossRefGoogle Scholar
  32. Tsai CS (1967) Spontaneous decarboxylation of oxalacetic acid. Can J Chem 45:873–880. doi:10.1139/v67-145 CrossRefGoogle Scholar
  33. Váli Z, Kilár F, Lakatos S, Venyaminov SA, Závodszky P (1980) L-Alanine dehydrogenase from Thermus thermophilus. Biochim Biophys Acta, Enzymol 615:34–47. doi:10.1016/0005-2744(80)90006-6 CrossRefGoogle Scholar
  34. Voellym R, Leisinger T (1976) Role of 4-aminobutyrate aminotransferase in the arginine metabolism of Pseudomonas aeruginosa. J Bacteriol 128:722–729PubMedPubMedCentralGoogle Scholar
  35. Wybenga GG, Crismaru CG, Janssen DB, Dijkstra BW (2012) Structural determinants of the β-selectivity of a bacterial aminotransferase. J Biol Chem 287:28495–28502. doi:10.1074/jbc.M112.375238 CrossRefPubMedPubMedCentralGoogle Scholar
  36. YASARA Structure, Version 14.7.17, YASARA Biosciences, 2014Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Fabian Steffen-Munsberg
    • 1
    • 2
  • Philipp Matzel
    • 3
  • Miriam A. Sowa
    • 3
  • Per Berglund
    • 2
  • Uwe T. Bornscheuer
    • 1
  • Matthias Höhne
    • 3
  1. 1.Department of Biotechnology & Enzyme Catalysis, Institute of BiochemistryGreifswald UniversityGreifswaldGermany
  2. 2.KTH Royal Institute of Technology, School of Biotechnology, Division of Industrial BiotechnologyAlbaNova University CenterStockholmSweden
  3. 3.Protein Biochemistry, Institute of BiochemistryGreifswald UniversityGreifswaldGermany

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