Skip to main content
SpringerLink
Log in

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

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • 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):252–256

    Article  CAS  PubMed  Google Scholar 

  • 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):452–455

    Article  CAS  PubMed  Google Scholar 

  • Beigi M, Waltzer S, Zarei M, Müller M (2014) New Stetter reactions catalyzed by thiamine diphosphate dependent MenD from E. coli. J Biotechnol 191:64–68

    Article  CAS  PubMed  Google Scholar 

  • 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 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brovetto M, Gamenara D, Méndez PS, Seoane GA (2011) C-C bond-forming lyases in organic synthesis. Chem Rev 111(7):4346–4403

    Article  CAS  PubMed  Google Scholar 

  • Busto E (2016) Recent developments in the preparation of carbohydrate derivatives from achiral building blocks by using aldolases. ChemCatChem 8(16):2589–2598

    Article  CAS  Google Scholar 

  • Clapés P, Fessner W-D, Sprenger GA, Samland AK (2010) Recent progress in stereoselective synthesis with aldolases. Curr Opin Chem Biol 14(2):154–167

    Article  PubMed  CAS  Google Scholar 

  • Clapés P, Garrabou X (2011) Current trends in asymmetric synthesis with aldolases. Adv Synth Catal 353(13):2263–2283

    Article  CAS  Google Scholar 

  • 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):99–107

    Article  CAS  Google Scholar 

  • Dresen C, Richter M, Pohl M, Lüdeke S, Müller M (2010) The enzymatic asymmetric conjugate umpolung reaction. Angew Chem Int Ed 49(37):6600–6603

    Article  CAS  Google Scholar 

  • 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):956–960

    Article  CAS  PubMed  Google Scholar 

  • Fesko K, Gruber-Khadjawi M (2013) Biocatalytic methods for C-C bond formation. ChemCatChem 5(6):1248–1272

    Article  CAS  Google Scholar 

  • 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):1085–1086

    Article  Google Scholar 

  • 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):555–557

    Article  CAS  Google Scholar 

  • 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):4441–4459

    Article  CAS  Google Scholar 

  • 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 359:2061–2065

    Article  CAS  Google Scholar 

  • 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 53:5465–5468

    Article  Google Scholar 

  • 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 280:6374–6394

    Article  CAS  PubMed  Google Scholar 

  • 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 21:3335–3346

    Article  CAS  PubMed  Google Scholar 

  • Hochuli E, Döbeli H, Schacher A (1987) New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr A 411:177–184

    Article  CAS  Google Scholar 

  • Hubrich F, Müller M, Andexer JN (2014) In vitro production and purification of isochorismate using a two-enzyme cascade. J Biotechnol 191:93–98

    Article  CAS  PubMed  Google Scholar 

  • 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):10979–10989

    Article  CAS  PubMed  Google Scholar 

  • Jordan F (2003) Current mechanistic understanding of thiamin diphosphate-dependent enzymatic reactions. Nat Prod Rep 20(2):184–201

    Article  CAS  PubMed  Google Scholar 

  • 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):9681–9690

    Article  CAS  PubMed  Google Scholar 

  • 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 213:757–767

    CAS  PubMed  Google Scholar 

  • 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 2:1580–1589

    Article  CAS  Google Scholar 

  • Kurutsch A, Richter M, Brecht V, Sprenger GA, Müller M (2009) MenD as a versatile catalyst for asymmetric synthesis. J Mol Catal B Enzym 61:56–66

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685

    Article  CAS  PubMed  Google Scholar 

  • 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):1204–1221

    Article  CAS  Google Scholar 

  • Müller M, Sprenger GA, Pohl M (2013) C–C bond formation using ThDP-dependent lyases. Curr Opin Chem Biol 17(2):261–270

    Article  PubMed  CAS  Google Scholar 

  • 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):1607–1625

    Article  CAS  PubMed  Google Scholar 

  • 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 44:171–181

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pohl M, Sprenger GA, Müller M (2004) A new perspective on thiamine catalysis. Curr Opin Biotechnol 15(4):335–342

    Article  CAS  PubMed  Google Scholar 

  • Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258(5536):598–599

    Article  CAS  PubMed  Google Scholar 

  • 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):13992–14006

    Article  CAS  PubMed  Google Scholar 

  • 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):e26021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Samland AK, Sprenger GA (2006) Microbial aldolases as C-C bonding enzymes – unknown treasures and new developments. Appl Microbiol Biotechnol 71(3):253–264

    Article  CAS  PubMed  Google Scholar 

  • Schmidt NG, Eger E, Kroutil W (2016) Building bridges: biocatalytic C-C-bond formation toward multifunctional products. ACS Catal 6(7):4286–4311

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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):11055–11061

    Article  PubMed  Google Scholar 

  • 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 159:188–194

    Article  CAS  PubMed  Google Scholar 

  • 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 273:70–77

    Article  CAS  PubMed  Google Scholar 

  • Sprenger GA (1995) Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12. Arch Microbiol 164(5):324–330

    Article  CAS  PubMed  Google Scholar 

  • 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 94:12857–12862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Studier FW (1991) Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. J Mol Biol 219(1):37–44

    Article  CAS  PubMed  Google Scholar 

  • 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 268:71–80

    Article  CAS  PubMed  Google Scholar 

  • Sukumaran J, Hanefeld U (2005) Enantioselective C-C bond synthesis catalysed by enzymes. Chem Soc Rev 34:530–542

    Article  CAS  PubMed  Google Scholar 

  • Walters MJ, Toone EJ (2007) Pyruvate aldolases in chiral carbon-carbon bond formation. Nat Protoc 2(7):1825–1830

    Article  CAS  PubMed  Google Scholar 

  • 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 16:710–720

    Article  CAS  PubMed  Google Scholar 

  • 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):3587–3594

    Article  CAS  Google Scholar 

  • 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):2061–2063

    Article  CAS  Google Scholar 

  • 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 6:1082–1088

    Article  CAS  Google Scholar 

  • Windle CL, Müller M, Nelson A, Berry A (2014) Engineering aldolases as biocatalysts. Curr Opin Chem Biol 19:25–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zimmermann FT, Schneider A, Schörken U, Sprenger GA, Fessner W-D (1999) Efficient multi-enzymatic synthesis of D-xylulose 5-phosphate. Tetrahedron Asymmetry 10:1643–1646

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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

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

Author information

Authors and Affiliations

  1. Institute of Microbiology, Universität Stuttgart, Allmandring 31, 70569, Stuttgart, Germany

    Matthias Schapfl, Shiromi Baier & Georg A. Sprenger

  2. Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany

    Alexander Fries, Sascha Ferlaino, Simon Waltzer & Michael Müller

Authors
  1. Matthias Schapfl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shiromi Baier
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexander Fries
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sascha Ferlaino
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Simon Waltzer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Georg A. Sprenger
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Georg A. Sprenger.

Ethics declarations

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.

Electronic supplementary material

ESM 1

(PDF 3523 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schapfl, M., Baier, S., Fries, A. et al. Extended substrate range of thiamine diphosphate-dependent MenD enzyme by coupling of two C–C-bonding reactions. Appl Microbiol Biotechnol 102, 8359–8372 (2018). https://doi.org/10.1007/s00253-018-9259-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-018-9259-z

Keywords

Search

Navigation