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Free keywords:
Models, Molecular
Molecular Structure
Oxidoreductases Acting on CH-CH Group Donors/chemistry/*metabolism
Polyketides/chemistry/*metabolism
*Protein Engineering
CO2 fixation
biotechnology
crotonyl-CoA carboxylase/reductase
natural product engineering
polyketides
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 alpha,beta-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 alpha-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.
