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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. Sprenger
Biotechnologically relevant enzymes and proteins
  • 135 Downloads

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.

Keywords

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

Notes

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 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)

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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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