Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes

Appl Microbiol Biotechnol (2014) 98:96819690 DOI 10.1007/s00253-014-5850-0

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

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 Mller

Received: 2 March 2014 /Revised: 21 May 2014 /Accepted: 22 May 2014 /Published online: 24 June 2014 # Springer-Verlag Berlin Heidelberg 2014

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 ,-unsaturated ketones (Michael acceptor substrates) and -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 carboncarbon bond formation reactions of aldehydes and -keto acids, resulting in 2-hydroxy ketones.

Keywords 1,4-Carboligation . Biocatalysis . CC coupling . Chemoenzymatic synthesis . Umpolung

Introduction

Stetter and Schreckenberg (1973) described the reaction of aromatic aldehydes (donor substrates) with ,-unsaturated carbonyl compounds (Michael acceptor substrates) in the presence of cyanide ions, which resulted in the formation of 1,4-addition products. Extension of this so-called Stetter reaction to heterocyclic aldehydes followed in 1976. The reaction scope for the intra- and intermolecular versions was improved with the application of thiazolium salts as catalysts, allowing for the CC bond coupling of aliphatic aldehydes (Stetter 1976).

Stereoselective intermolecular Stetter reactions are gaining increasing interest in asymmetric synthesis. Chiral thiazolium and triazolium salts as enantioselective catalysts...