Applied Microbiology and Biotechnology

, Volume 98, Issue 23, pp 9681–9690 | Cite as

Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

  • Elena Kasparyan
  • Michael Richter
  • Carola Dresen
  • Lydia S. Walter
  • Georg Fuchs
  • Finian J. Leeper
  • Tobias Wacker
  • Susana L. A. Andrade
  • Geraldine Kolter
  • Martina Pohl
  • Michael Müller
Biotechnologically relevant enzymes and proteins

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.

Keywords

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

Supplementary material

253_2014_5850_MOESM1_ESM.pdf (247 kb)
ESM 1(PDF 246 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Elena Kasparyan
    • 1
  • Michael Richter
    • 2
  • Carola Dresen
    • 1
  • Lydia S. Walter
    • 1
  • Georg Fuchs
    • 3
  • Finian J. Leeper
    • 4
  • Tobias Wacker
    • 5
  • Susana L. A. Andrade
    • 5
  • Geraldine Kolter
    • 6
  • Martina Pohl
    • 6
  • Michael Müller
    • 1
  1. 1.Institute of Pharmaceutical SciencesAlbert-Ludwigs-Universität FreiburgFreiburgGermany
  2. 2.Laboratory for Biomaterials, EmpaSwiss Federal Laboratories for Materials Science and TechnologySt. GallenSwitzerland
  3. 3.Institute of Biology IIAlbert-Ludwigs-Universität FreiburgFreiburgGermany
  4. 4.Department of ChemistryUniversity of CambridgeCambridgeUK
  5. 5.Institute of BiochemistryAlbert-Ludwigs-Universität FreiburgFreiburgGermany
  6. 6.IBG-1: BiotechnologyForschungszentrum Jülich GmbHJülichGermany

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