5/17/2023 0 Comments Enzymex pcTo improve further the carboxylation efficiency of PCS EN, we compared the active site of PCS EN (PDB: 6EQO) with ECR Ks. Carboxylation yields are calculated from mean carboxylation yields over five time points in three replicates. K M values were determined from a Michaelis–Menten fit of at least 18 data points, with fixed acrylyl-CoA concentrations for K M_CO2 ( Figures S3 and S4, Table S2 for k cat. shows combined reduction and carboxylation activity. Notably, the carboxylation function was not limited to the Erythrobacter enzyme, but was also detected with PCS from Chloroflexus aurantiacus (PCS Ca, Table S1). Incorporation of 13CO 2-label confirmed the latent carboxylation activity of PCS EN ( Figure 2b). NAP1, PCS EN, at 4.4 mM dissolved CO 2, we detected minor amounts of methylmalonyl-CoA besides the main product propionyl-CoA. ![]() (11) When we assayed PCS from Erythrobacter sp. The enzyme forms a central reaction chamber, in which three subsequent reactions take place in a synchronized fashion. PCS is a three-domain fusion enzyme that catalyzes the overall conversion of 3-hydroxypropionate to propionyl-CoA (10) ( Figure 2a). We decided to test selected members of these enzyme families in their CO 2-fixing capabilities. The AER family is more distantly related to the ECR family, and selected homologues only contain one or two of the four conserved residues of the CO 2-binding pocket ( Figure S2). The PCS family clusters closely to ECRs and shows a fully conserved CO 2-binding motif across individual family members ( Figure S1). Our search revealed two enzyme families that show the potential to bind CO 2, the propionyl-CoA synthase (PCS) and an archaeal enoyl-CoA reductase (AER) family ( Figure 1b). To identify enzyme scaffolds capable of binding CO 2 beyond the ECR enzyme family, we searched homologues of the MDR superfamily for the CO 2-binding motif. Our results provide a strategy to develop novel CO 2-fixing enzymes and shed light on the emergence of natural carboxylases during evolution. The engineered carboxylases show improved CO 2-binding and kinetic parameters comparable to naturally existing CO 2-fixing enzymes. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO 2 and suppressing diffusion of water to the active site. ![]() NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We show that propionyl-CoA synthase from Erythrobacter sp. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. Here we used bioinformatics to identify a “sleeping carboxylase function” in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. Developing new carbon dioxide (CO 2) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology.
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