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Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

Applied Microbiology and Biotechnology volume 98, pages 9681–9690 (2014)Cite this article

Abstract

The intermolecular asymmetric Stetter reaction is an almost unexplored transformation for biocatalysts. Previously reported thiamine diphosphate (ThDP)-dependent PigD from Serratia marcescens is the first enzyme identified to catalyze the Stetter reaction of α,β-unsaturated ketones (Michael acceptor substrates) and α-keto acids. PigD is involved in the biosynthesis of the potent cytotoxic agent prodigiosin. Here, we describe the investigation of two new ThDP-dependent enzymes, SeAAS from Saccharopolyspora erythraea and HapD from Hahella chejuensis. Both show a high degree of homology to the amino acid sequence of PigD (39 and 51 %, respectively). The new enzymes were heterologously overproduced in Escherichia coli, and the yield of soluble protein was enhanced by co-expression of the chaperone genes groEL/ES. SeAAS and HapD catalyze intermolecular Stetter reactions in vitro with high enantioselectivity. The enzymes possess a characteristic substrate range with respect to Michael acceptor substrates. This provides support for a new type of ThDP-dependent enzymatic activity, which is abundant in various species and not restricted to prodigiosin biosynthesis in different strains. Moreover, PigD, SeAAS, and HapD are also able to catalyze asymmetric carbon–carbon bond formation reactions of aldehydes and α-keto acids, resulting in 2-hydroxy ketones.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the scope of the Research Group FOR 1296. We are grateful to Prof. Peter Leadlay, University of Cambridge, and Dr. Wolfgang Hüttel, Albert-Ludwigs-Universität Freiburg, for providing plasmid pL1SL2 and for helpful discussions. The technical assistance of Fabrizio Bonina, Volker Brecht, Wolfgang Kornberger, and Simon Waltzer, Albert-Ludwigs-Universität Freiburg, is gratefully acknowledged. We thank Dr. Mostafa Zarei, Center for Biological Systems Analysis, Freiburg, for MS determinations.

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Authors and Affiliations

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

    Elena Kasparyan, Carola Dresen, Lydia S. Walter & Michael Müller

  2. Laboratory for Biomaterials, Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, 9014, St. Gallen, Switzerland

    Michael Richter

  3. Institute of Biology II, Albert-Ludwigs-Universität Freiburg, Schänzlestrasse 1, 79104, Freiburg, Germany

    Georg Fuchs

  4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK

    Finian J. Leeper

  5. Institute of Biochemistry, Albert-Ludwigs-Universität Freiburg, Albertstrasse 19, 79104, Freiburg, Germany

    Tobias Wacker & Susana L. A. Andrade

  6. IBG-1: Biotechnology, Forschungszentrum Jülich GmbH, 52425, Jülich, Germany

    Geraldine Kolter & Martina Pohl

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  1. Elena Kasparyan
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Correspondence to Michael Müller.

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Kasparyan, E., Richter, M., Dresen, C. et al. Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes. Appl Microbiol Biotechnol 98, 9681–9690 (2014). https://doi.org/10.1007/s00253-014-5850-0

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  • DOI: https://doi.org/10.1007/s00253-014-5850-0

Keywords

  • 1,4-Carboligation
  • Biocatalysis
  • C–C coupling
  • Chemoenzymatic synthesis
  • Umpolung