Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis. Issue 45 (18th September 2015)

• Record Type: Journal Article
• Authors: Peter, Dominik M.; Schada von Borzyskowski, Lennart; Kiefer, Patrick; Christen, Philipp; Vorholt, Julia A.; Erb, Tobias J.
• Is Part Of: Angewandte Chemie international edition. Volume 54:Issue 45(2015)
• Page Start: 13457
• Page End: 13461
• Publication Date: 2015-09-18
• Subjects: biotechnology -- CO2 fixation -- crotonyl‐CoA carboxylase/reductase -- natural product engineering -- polyketides; Chemistry -- Periodicals; 540
• DOI: 10.1002/anie.201505282
• Languages: English
• ISSNs: 1433-7851

Abstract

Abstract: Carboxylating enoyl‐thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α, β‐unsaturated CoA‐thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl‐CoA during assimilation of the central metabolite acetyl‐CoA. In secondary metabolism they provide distinct α‐carboxyl‐acyl‐thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5‐enoyl‐CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof‐of‐principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification. Abstract : Engineering promiscuity : The molecular basis for substrate specificity of carboxylating enoyl‐CoA reductases was identified by screening a CoA‐thioester substrate library against a library of enzyme homologues. Site‐directed mutagenesis of the promiscuity‐determining sites enabled a previously restrictive enzyme to accept a variety of new, bulky substrates.