New oxidoreductases from cyanobacteria: Exploring nature's diversity

Abstract

The need for new and efficient biocatalysts for asymmetric synthesis is caused by the increasing demand for enantiopure chiral alcohols in the chemical and pharmaceutical industries. One approach to find new enzymes for technical applications is the screening of biodiversity. The identification of a novel oxidoreductase in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942, which is capable of enantioselectively reducing sterically demanding and multi-halogenated ketones, led to a search for homologous enzymes in other cyanobacterial strains. Here, we present a comparative study of 3-ketoacyl-(acyl-carrier-protein)-reductases from 16 cyanobacteria belonging to different taxonomic groups. The genes encoding the respective oxidoreductases were cloned and expressed in Escherichia coli. The purified enzymes were used for the reduction of four exemplarily chosen prochiral ketones. In either case, the specific enzymatic activities differed by two to three orders of magnitude. For example, ethyl 4-chloroacetoacetate was reduced with 0.1–250 U mg −1 to the corresponding ( S)-alcohol in moderate to excellent enantiomeric excesses (53.4% to >99.8%). The 3-ketoacyl-(acyl-carrier-protein) reductase from the marine cyanobacterium Synechococcus sp. strain RCC 307 showed a higher activity with all of the four substrates under study compared to the originally described biocatalyst from Synechococcus sp. strain PCC 7942. The reaction rates were up to 323% higher with comparable or better enantioselectivity. The analysis of relationships between protein sequences and enzyme characteristics led to the discovery of amino acid residues influencing catalytic properties of cyanobacterial KRs and thus provide the basis for a subsequent rational protein design.