Applied Microbiology and Biotechnology

, Volume 99, Issue 22, pp 9651–9661 | Cite as

Expanding the threonine aldolase toolbox for the asymmetric synthesis of tertiary α-amino acids

  • Kateryna Fesko
  • Gernot A. Strohmeier
  • Rolf Breinbauer
Biotechnologically relevant enzymes and proteins


The direct biochemical synthesis of tertiary α-amino acids with a wide range of diversity was recently reported using natural threonine aldolases LTA from Aeromonas jandei and DTA from Pseudomonas sp. Here, we describe the identification of five novel threonine aldolases which accept alanine and serine as amino acid donors. The enzymes were found by sequence database analysis using known aldolases as template. All enzymes were overexpressed in Escherichia coli and purified, and their biochemical properties were characterized. The new enantiocomplementary l- and d-threonine aldolases catalyze the asymmetric synthesis of β-hydroxy α-methyl- and α-hydroxymethyl-α-amino acids with good conversion and perfect enantioselectivity at α-carbon of the products (e.e. >99 %). The structural basis for the broad donor specificity of these threonine aldolases is analyzed based on crystal structure alignments and amino acid sequences comparison.


Threonine aldolase Aldol reactions Tertiary amino acids Enzyme catalysis Biocatalysis 



The activity leading to the present results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contribution for the Innovative Medicine Initiative under Grant Agreement No. 115360 (Chemical manufacturing methods for the 21st century pharmaceutical industries, CHEM21). We would like to thank Dr. Martina Geier for the support with genetic experiments.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was obtained from all individual participants included in the study.

Conflict of interest

The authors declare that they have no competing interests.


  1. Avenoza A, Busto JH, Corzana F, Peregrina JM, Sucunza D, Zurbano MM (2004) α-Methylserinals as an access to α-methyl-β-hydroxy amino acids: application in the synthesis of all stereoisomers of α-methylthreonine. Tetrahedron Asymmetry 15:719–724CrossRefGoogle Scholar
  2. Baker P, Seah SYK (2012) Rational approaches for engineering novel functionalities in carbon-carbon bond forming enzymes. Comput Struct Biotechnol J 2:1–10CrossRefGoogle Scholar
  3. Bornscheuer UT, Kazlauskas RJ (2004) Catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew Chem Int Ed 43:6032–6040CrossRefGoogle Scholar
  4. Clapes P, Garrabou X (2011) Current trends in asymmetric synthesis with aldolases. Adv Synth Catal 353:2263–2283CrossRefGoogle Scholar
  5. Davids T, Schmidt M, Böttcher D, Bornscheuer UT (2013) Strategies for the discovery and engineering of enzymes for biocatalysis. Curr Opin Chem Biol 17:215–220CrossRefPubMedGoogle Scholar
  6. di Salvo ML, Soumya G, Remesh M, Vivoli M, Ghatge MS, Paiardini A, D’Aguanno S, Safo MK, Contestabile R (2014) On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase. FEBS J 281:129–145CrossRefPubMedGoogle Scholar
  7. Dietz F, Gröger H (2009) Asymmetric synthesis of all stereoisomers of α-methylthreonine using an organocatalytic steglich rearrangement reaction as a key step. Synlett 24:4208–4218Google Scholar
  8. Dückers N, Baer K, Simon S, Gröger H, Hummel W (2010) Threonine aldolases -screening, properties and applications in the synthesis of non-proteinogenic β-hydroxy-α-amino acids. Appl Microbiol Biotechnol 88:409–424CrossRefPubMedGoogle Scholar
  9. Fesko K, Gruber M (2013) Biocatalytic methods for C-C bond formation. ChemCatChem 5:1248CrossRefGoogle Scholar
  10. Fesko K, Reisinger C, Steinreiber J, Weber H, Schürmann M, Griengl H (2008) Four types of threonine aldolases: similarities and differences in kinetics/thermodynamics. J Mol Catal B 52–53:19–26CrossRefGoogle Scholar
  11. Fesko K, Uhl M, Steinreiber J, Gruber K, Griengl H (2010) Biocatalytic access to α, α-dialkyl-α-amino acids by a mechanism-based approach. Angew Chem Int Ed 49:121–124CrossRefGoogle Scholar
  12. Fessner WD (2011) Aldol reactions. In: Drauz K, Gröger H, May O (eds) Enzyme catalysis in organic synthesis, 3rd edn. Wiley-VCH, Weinheim, pp 857–917Google Scholar
  13. Gatti-Lafranconi P, Hollfelder F (2013) Flexibility and reactivity in promiscuous enzymes. ChemBioChem 14:285–292CrossRefPubMedGoogle Scholar
  14. Grandel R, Kazmaier U (1998) Diastereoselective synthesis of β-substituted α-methylserines via alanine ester enolates. Eur J Org Chem 2:409–417Google Scholar
  15. Hernandez K, Zelen I, Petrillo G, Uson I, Wandtke CM, Bujons J, Joglar J, Parella T, Clapes P (2015) Engineered L-serine hydroxymethyltransferase from Streptococcus thermophilius for the synthesis of α, α-dialkyl-α-amino acids. Angew Chem Int Ed 54:3013–3017CrossRefGoogle Scholar
  16. Kataoka M, Wada M, Nishi K, Yamada H, Shimizu S (1997) Purification and characterization of L-allo-threonine aldolase from Aeromonas jandaei DK-39. FEMS Microbiol Lett 151(2):245–248CrossRefPubMedGoogle Scholar
  17. Kielkopf CL, Burley SK (2002) X-ray structures of threonine aldolase complexes: structural basis of substrate recognition. Biochemistry 41:11711–11720CrossRefPubMedGoogle Scholar
  18. Müller M (2012) Recent developments in enzymatic asymmetric C-C bond formation. Adv Synth Catal 354:3161–3174CrossRefGoogle Scholar
  19. Qin HM, Imai FL, Miyakawa T, Kataoka M, Okai M, Ohtsuka J, Hou F, Nagata K, Shimizu S, Tanokura M (2014) L-allo-Threonine aldolase with an H128Y/S292R mutation from Aeromonas jandaei DK-39 reveals the structural basis of changes in substrate stereoselectivity. Acta Crystallogr Sect D 70:1695–1703CrossRefGoogle Scholar
  20. Reisinger C, Kern A, Fesko K, Schwab H (2007) An efficient plasmid vector for expression cloning of large numbers of PCR fragments in Escherichia coli. Appl Microbiol Biotechnol 77:241–244CrossRefPubMedGoogle Scholar
  21. Steinreiber J, Fesko K, Reisinger C, Schürmann M, Assema F, Wolberg M, Mink D, Griengl H (2007a) Threonine aldolases - an emerging tool for organic synthesis. Tetrahedron 63:918–926CrossRefGoogle Scholar
  22. Steinreiber J, Fesko K, Reisinger C, Schürmann M, Assema F, Griengl H (2007b) Synthesis of γ-halogenated and long-chain β-hydroxy-α-amino acids and 2-amino-1,3-diols using threonine aldolases. Tetrahedron 63:8088–8093CrossRefGoogle Scholar
  23. Toney MD (2005) Reaction specificity in pyridoxal phosphate enzymes. Arch Biochem Biophys 433:279–287CrossRefPubMedGoogle Scholar
  24. Vogt H, Bräse S (2007) Recent approaches towards the asymmetric synthesis of α, α-disubstituted α-amino acids. Org Biomol Chem 5:406–430CrossRefPubMedGoogle Scholar
  25. Wildmann M, Pleiss J, Samland AK (2012) Computational tools for rational protein design of aldolases. Comput Struct Biotechnol J 2:1–11CrossRefGoogle Scholar
  26. Windle C, Müller M, Nelson A, Berry A (2014) Engineering aldolases as biocatalysts. Curr Opin Chem Biol 19:25–33CrossRefPubMedPubMedCentralGoogle Scholar
  27. Zhang WH, Otting G, Jackson CJ (2013) Protein engineering with unnatural amino acids. Curr Opin Struct Biol 23:581–587CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Kateryna Fesko
    • 1
  • Gernot A. Strohmeier
    • 1
    • 2
  • Rolf Breinbauer
    • 1
    • 2
  1. 1.Institute of Organic ChemistryGraz University of TechnologyGrazAustria
  2. 2.Austrian Centre of Industrial Biotechnology (ACIB) GmbHGrazAustria

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