Applied Microbiology and Biotechnology

, Volume 102, Issue 19, pp 8359–8372 | Cite as

Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C–C-bonding reactions

  • Matthias Schapfl
  • Shiromi Baier
  • Alexander Fries
  • Sascha Ferlaino
  • Simon Waltzer
  • Michael Müller
  • Georg A. SprengerEmail author
Biotechnologically relevant enzymes and proteins


Carboligations catalyzed by aldolases or thiamine diphosphate (ThDP)-dependent enzymes are well-known in biocatalysis to deliver enantioselective chain elongation reactions. A pyruvate-dependent aldolase (2-oxo-3-deoxy-6-phosphogluconate aldolase [EDA]) introduces a chiral center when reacting with the electrophile, glyoxylic acid, delivering the (S)-enantiomer of (4S)-4-hydroxy-2-oxoglutarate [(S)-HOG]. The ThDP-dependent enzyme MenD (2-succinyl-5-enol-pyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (SEPHCHC synthase)) enables access to highly functionalized substances by forming intermolecular C–C bonds with Michael acceptor compounds by a Stetter-like 1,4- or a benzoin-condensation 1,2-addition of activated succinyl semialdehyde (ThDP adduct formed by decarboxylation of 2-oxoglutarate). MenD-catalyzed reactions are characterized by high chemo- and regioselectivity. Here, we report (S)-HOG, in situ formed by EDA, to serve as new donor substrate for MenD in 1,4-addition reactions with 2,3-trans-CHD (2,3-trans-dihydroxy-cyclohexadiene carboxylate) and acrylic acid. Likewise, (S)-HOG serves as donor in 1,2-additions with aromatic (benzaldehyde) and aliphatic (hexanal) aldehydes. This enzyme cascade of two subsequent C–C bond formations (EDA aldolase and a ThDP-dependent carboligase, MenD) generates two new stereocenters.


Aldolase EDA ThDP-dependent enzyme MenD 4-hydroxy-2-oxoglutarate Carboligations Stetter-like 1,4-additions 1,2-additions 



The authors thank Dr. Bernd Nebel, University of Stuttgart, Department of Technical Biochemistry, Institute for Biochemistry and Technical Biochemistry, for the assistance with LC-MS measurements, data handling, and valuable discussions. We thank Martina Pohl (FZ Jülich) for the (S)-selective MenD variant and Steffen Lüdecke and Marija Marolt (University of Freiburg) for CD measurements of HOG.

Funding information

We thank the German Research Foundation DFG for the financial support within the framework of project FOR1296 “Diversity of asymmetric thiamine catalysis.”

Compliance with ethical standards

Conflict of interest

The authors declare that they have 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_2018_9259_MOESM1_ESM.pdf (3.4 mb)
ESM 1 (PDF 3523 kb)


  1. Beigi M, Loschonsky S, Lehwald P, Brecht V, Andrade SLA, Leeper FJ, Hummel W, Müller M (2013a) α-Hydroxy-β-keto acid rearrangement–decarboxylation: impact on ThDP-dependent enzymatic transformations. Org Biomol Chem 11(2):252–256CrossRefPubMedGoogle Scholar
  2. Beigi M, Waltzer S, Fries A, Eggeling L, Sprenger GA, Müller M (2013b) TCA cycle involved enzymes SucA and Kgd, as well as MenD: efficient biocatalysts for asymmetric C–C bond formation. Org Lett 15(3):452–455CrossRefPubMedGoogle Scholar
  3. Beigi M, Waltzer S, Zarei M, Müller M (2014) New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli. J Biotechnol 191:64–68CrossRefPubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brovetto M, Gamenara D, Méndez PS, Seoane GA (2011) C-C bond-forming lyases in organic synthesis. Chem Rev 111(7):4346–4403CrossRefPubMedGoogle Scholar
  6. Busto E (2016) Recent developments in the preparation of carbohydrate derivatives from achiral building blocks by using aldolases. ChemCatChem 8(16):2589–2598CrossRefGoogle Scholar
  7. Clapés P, Fessner W-D, Sprenger GA, Samland AK (2010) Recent progress in stereoselective synthesis with aldolases. Curr Opin Chem Biol 14(2):154–167CrossRefPubMedGoogle Scholar
  8. Clapés P, Garrabou X (2011) Current trends in asymmetric synthesis with aldolases. Adv Synth Catal 353(13):2263–2283CrossRefGoogle Scholar
  9. Climent MJ, Corma A, Iborra S, Mifsud M, Velty A (2010) New one-pot multistep process with multifunctional catalysts. Decreasing the E factor in the synthesis of fine chemicals. Green Chem 12(1):99–107CrossRefGoogle Scholar
  10. Dresen C, Richter M, Pohl M, Lüdeke S, Müller M (2010) The enzymatic asymmetric conjugate umpolung reaction. Angew Chem Int Ed 49(37):6600–6603CrossRefGoogle Scholar
  11. Emmons GT, Campbell IM, Bentley R (1985) Vitamin K (menaquinone) biosynthesis in bacteria. Purification and probable structure of an intermediate prior to o-succinylbenzoate. Biochem Biophys Res Commun 131(2):956–960CrossRefPubMedGoogle Scholar
  12. Fesko K, Gruber-Khadjawi M (2013) Biocatalytic methods for C-C bond formation. ChemCatChem 5(6):1248–1272CrossRefGoogle Scholar
  13. Floyd NC, Liebster MH, Turner NJ (1992) A simple strategy for obtaining both enantiomers from an aldolase reaction. Preparation of L- and D-4-hydroxy-2-ketoglutarate. J Chem Soc Perkin Trans 1(9):1085–1086CrossRefGoogle Scholar
  14. Franke D, Sprenger GA, Müller M (2001) Synthesis of functionalized cyclohexadiene-trans-diols with recombinant cells of Escherichia coli. Angew Chem Int Ed 40(3):555–557CrossRefGoogle Scholar
  15. Giovannini PP, Bortolini O, Massi A (2016) Thiamine-diphosphate-dependent enzymes as catalytic tools for the asymmetric benzoin-type reaction. Eur J Org Chem 2016(26):4441–4459CrossRefGoogle Scholar
  16. Guérard-Hélaine C, Lopes Moreira MDS, Touisni N, Hecquet L, Lemaire M, Hélaine V (2017a) Transketolase-aldolase symbiosis for the stereoselective preparation of aldoles and ketoses of biological interest. Adv Synth Catal 359:2061–2065CrossRefGoogle Scholar
  17. Guérard-Hélaine C, Heuson E, Ndiaye M, Gourbeyre L, Lemaire M, Hélaine V, Charmantray F, Petit J-L, Salanoubat M, de Berardinis V, Gefflaut T (2017b) Stereoselective synthesis fo γ-hydroxy-α-amino acids through aldolase-transaminase recycling cascades. Chem Commun 53:5465–5468CrossRefGoogle Scholar
  18. Hailes H, Rother D, Müller M, Westphal R, Ward J, Pleiss P, Vogel C, Pohl M (2013) Engineering stereoselectivity of ThDP-dependent enzymes. FEBS J 280:6374–6394CrossRefPubMedGoogle Scholar
  19. Hernandez K, Parella T, Joglar J, Bujons J, Pohl M, Clapés P (2015) Expedient synthesis of C-aryl carbohydrates by consecutive biocatalytic benzoin and aldol reactions. Chem Eur J 21:3335–3346CrossRefPubMedGoogle Scholar
  20. Hochuli E, Döbeli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 411:177–184CrossRefGoogle Scholar
  21. Hubrich F, Müller M, Andexer JN (2014) In vitro production and purification of isochorismate using a two-enzyme cascade. J Biotechnol 191:93–98CrossRefPubMedGoogle Scholar
  22. Jiang M, Cao Y, Guo ZF, Chen M, Chen X, Guo Z (2007) Menaquinone biosynthesis in Escherichia coli. Identification of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate as a novel intermediate and re-evaluation of MenD activity. Biochemistry 46(38):10979–10989CrossRefPubMedGoogle Scholar
  23. Jordan F (2003) Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions. Nat Prod Rep 20(2):184–201CrossRefPubMedGoogle Scholar
  24. Kasparyan E, Richter M, Dresen C, Walter LS, Fuchs G, Leeper FJ, Wacker T, Andrade SLA, Kolter G, Pohl M, Müller M (2014) Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes. Appl Microbiol Biotechnol 98(23):9681–9690CrossRefPubMedGoogle Scholar
  25. Kovachevich R, Wood WA (1955) Carbohydrate metabolism by Pseudomonas fluorescens. IV. Purification and properties of 2-keto-3-deoxy-6-phosphogluconate aldolase. J Biol Chem 213:757–767PubMedGoogle Scholar
  26. Kulig J, Simon RC, Rose CA, Husain SM, Häckh M, Lüdeke S, Zeitler K, Kroutil W, Pohl M, Rother D (2012) Stereoselective synthesis of bulky 1,2-diols with alcohol dehydrogenases. Catal Sci Technol 2:1580–1589CrossRefGoogle Scholar
  27. Kurutsch A, Richter M, Brecht V, Sprenger GA, Müller M (2009) MenD as a versatile catalyst for asymmetric synthesis. J Mol Catal B Enzym 61:56–66CrossRefGoogle Scholar
  28. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685CrossRefPubMedPubMedCentralGoogle Scholar
  29. López-Iglesias M, Méndez-Sánchez D, Gotor-Fernández V (2016) Native proteins in organic chemistry. Recent achievements in the use of non hydrolytic enzymes for the synthesis of pharmaceuticals. Curr Org Chem 20(11):1204–1221CrossRefGoogle Scholar
  30. Müller M, Sprenger GA, Pohl M (2013) C–C bond formation using ThDP-dependent lyases. Curr Opin Chem Biol 17(2):261–270CrossRefPubMedGoogle Scholar
  31. Ogawa J, Yamanaka H, Mano J, Doi M, Horinouchi N, Kodera T, Nio N, Smirnov SV, Samsonova NN, Kozlova YI, Shimizu S (2007) Synthesis of 4-hydroxyisoleucine by the aldolase-transaminase from Arthrobacter simplex AKU 626. Biosci Biotechnol Biochem 71(7):1607–1625CrossRefPubMedGoogle Scholar
  32. Pertusi DA, Moura ME, Jeffryes JG, Prabhu S, Biggs BW, Tyo KEJ (2017) Predicting novel substrates for enzymes with minimal experimental effort with active learning. Metab Eng 44:171–181CrossRefPubMedGoogle Scholar
  33. Pohl M, Sprenger GA, Müller M (2004) A new perspective on thiamine catalysis. Curr Opin Biotechnol 15(4):335–342CrossRefPubMedGoogle Scholar
  34. Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598–599CrossRefPubMedGoogle Scholar
  35. Prier CK, Arnold FH (2015) Chemomimetic biocatalysis: exploiting the synthetic potential of cofactor-dependent enzymes to create new catalysts. J Am Chem Soc 137(44):13992–14006CrossRefPubMedGoogle Scholar
  36. Riedel TJ, Johnson LC, Knight J, Hantgan RR, Holmes RP, Lowther WT (2011) Structural and biochemical studies of human 4-hydroxy-2-oxoglutarate aldolase: implications for hydroxyproline metabolism in primary hyperoxaluria. PLoS One 6(10):e26021CrossRefPubMedPubMedCentralGoogle Scholar
  37. Samland AK, Sprenger GA (2006) Microbial aldolases as C-C bonding enzymes – unknown treasures and new developments. Appl Microbiol Biotechnol 71(3):253–264CrossRefPubMedGoogle Scholar
  38. Schmidt NG, Eger E, Kroutil W (2016) Building bridges: biocatalytic C-C-bond formation toward multifunctional products. ACS Catal 6(7):4286–4311CrossRefPubMedPubMedCentralGoogle Scholar
  39. Schürmann M, Sprenger GA (2001) Fructose-6-phosphate aldolase is a novel class I aldolase from Escherichia coli and is related to a novel group of bacterial transaldolases. J Biol Chem 276(14):11055–11061CrossRefPubMedGoogle Scholar
  40. Sehl T, Simon R, Hailes H, Ward J, Schell U, Pohl M, Rother D (2012) TTC-based screening assay for ω-transaminases: a rapid method to detect reduction of 2-hydroxy ketones. J Biotechnol 159:188–194CrossRefPubMedGoogle Scholar
  41. Smirnov SV, Samsonova NN, Novikova AE, Matrosov NG, Rushkevich NY, Kodera T, Ogawa J, Yamanaka H, Shimizu S (2007) A novel strategy for enzymatic synthesis of 4-hydroxyisoleucine: identification of an enzyme possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase activity. FEMS Microbiol Lett 273:70–77CrossRefPubMedGoogle Scholar
  42. Sprenger GA (1995) Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12. Arch Microbiol 164(5):324–330CrossRefPubMedGoogle Scholar
  43. Sprenger GA, Schörken U, Wiegert T, Grolle S, de Graaf AA, Taylor SV, Begley TP, Bringer-Meyer S, Sahm H (1997) Identification of a thiamin-dependent synthase in Escherichia coli required for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol. Proc Natl Acad Sci U S A 94:12857–12862CrossRefPubMedPubMedCentralGoogle Scholar
  44. Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219(1):37–44CrossRefPubMedGoogle Scholar
  45. Sudar M, Vasić-Rački D, Müller M, Findrik Z (2018) Mathematical model of the MenD-catalyzed 1,4-addition (Stetter reaction) of α-ketoglutaric acid to acrylonitrile. J Biotechnol 268:71–80CrossRefPubMedGoogle Scholar
  46. Sukumaran J, Hanefeld U (2005) Enantioselective C-C bond synthesis catalysed by enzymes. Chem Soc Rev 34:530–542CrossRefPubMedGoogle Scholar
  47. Walters MJ, Toone EJ (2007) Pyruvate aldolases in chiral carbon-carbon bond formation. Nat Protoc 2(7):1825–1830CrossRefPubMedGoogle Scholar
  48. Walters MJ, Srikannathasan V, McEwan AR, Naismith JH, Fierke CA, Toone EJ (2008) Characterization and crystal structure of Escherichia coli KDPGal aldolase. Bioorg Med Chem 16:710–720CrossRefPubMedGoogle Scholar
  49. Westphal R, Hahn D, Mackfeld U, Waltzer S, Beigi M, Widmann M, Vogel C, Pleiss J, Müller M, Rother D, Pohl M (2013a) Tailoring the S-selectivity of 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase (MenD) from Escherichia coli. ChemCatChem 5(12):3587–3594CrossRefGoogle Scholar
  50. Westphal R, Waltzer S, Mackfeld U, Widmann M, Pleiss J, Beigi M, Müller M, Rother D, Pohl M (2013b) (S)-selective MenD variants from Escherichia coli provide access to new functionalized chiral α-hydroxy ketones. Chem Commun 49(20):2061–2063CrossRefGoogle Scholar
  51. Westphal R, Jansen S, Vogel C, Pleiss J, Müller M, Rother D, Pohl M (2014) MenD from Bacillus subtilis: a potent catalyst for the enantiocomplementary asymmetric synthesis of functionalized α-hydroxy ketones. ChemCatChem 6:1082–1088CrossRefGoogle Scholar
  52. Windle CL, Müller M, Nelson A, Berry A (2014) Engineering aldolases as biocatalysts. Curr Opin Chem Biol 19:25–33CrossRefPubMedPubMedCentralGoogle Scholar
  53. Zimmermann FT, Schneider A, Schörken U, Sprenger GA, Fessner W-D (1999) Efficient multi-enzymatic synthesis of D-xylulose 5-phosphate. Tetrahedron Asymmetry 10:1643–1646CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Institute of MicrobiologyUniversität StuttgartStuttgartGermany
  2. 2.Institute of Pharmaceutical SciencesAlbert-Ludwigs-Universität FreiburgFreiburgGermany

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