title: Asymmetric Stetter reactions catalyzed by thiamine diphosphate-dependent enzymes.
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.
authors: Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstrasse 25, 79104, Freiburg, Germany.
authors: Elena
authors: E
authors: Kasparyan
authors: Michael
authors: M
authors: Richter
authors: Carola
authors: C
authors: Dresen
authors: Lydia S
authors: LS
authors: Walter
authors: Georg
authors: G
authors: Fuchs
authors: Finian J
authors: FJ
authors: Leeper
authors: Tobias
authors: T
authors: Wacker
authors: Susana L A
authors: SL
authors: Andrade
authors: Geraldine
authors: G
authors: Kolter
authors: Martina
authors: M
authors: Pohl
authors: Michael
authors: M
authors: Müller
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D002264:Carboxylic Acids / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D007659:Ketones / Q000378:metabolism*; D008969:Molecular Sequence Data; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D002264:Carboxylic Acids; D003067:Coenzymes; D004269:DNA, Bacterial; D004798:Enzymes; D007659:Ketones; D013835:Thiamine Pyrophosphate