Abstract
Developing new carbon dioxide (CO2) fixing enzymes is a prerequisite to create new biocatalysts for diverse applications in chemistry, biotechnology and synthetic biology. Here we used bioinformatics to identify a “sleeping carboxylase function” in the superfamily of medium-chain dehydrogenases/reductases (MDR), i.e. enzymes that possess a low carboxylation side activity next to their original enzyme reaction. We show that propionyl-CoA synthase from Erythrobacter sp. NAP1, as well as an acrylyl-CoA reductase from Nitrosopumilus maritimus possess carboxylation yields of 3 ± 1 and 4.5 ± 0.9%. We use rational design to engineer these enzymes further into carboxylases by increasing interactions of the proteins with CO2 and suppressing diffusion of water to the active site. The engineered carboxylases show improved CO2-binding and kinetic parameters comparable to naturally existing CO2-fixing enzymes. Our results provide a strategy to develop novel CO2-fixing enzymes and shed light on the emergence of natural carboxylases during evolution.
Highlights
For enoyl-CoA carboxylase/reductase from Kitasatospora setae (ECRKs), four conserved amino acids that form a CO2-binding pocket at the active site were described recently[7] (Figure 1a). These four amino acids anchor and position the CO2 molecule during catalysis, in which a reactive enolate is formed that attacks the CO2.8
The archaeal enoyl-CoA reductase (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 CO2-binding pocket (Figure S2)
The carboxylation function was not limited to the Erythrobacter enzyme, but was detected with propionyl-CoA synthase (PCS) from Chloroflexus aurantiacus (PCSCa, Table S1)
Summary
Communication mM dissolved CO2, the enzyme showed a carboxylation yield (defined as percentage yield of carboxylated product compared with total product formed, including reduced side product) of 3 ± 1% (Table 1). This showed that the reductase domain is able to carboxylate acrylyl-CoA directly. We directly tested the reductase domain for carboxylation activity with an E1027Q variant of PCSEN (PCSEN_ΔDH) that is unable to generate acrylyl-CoA. Locking Asn1301 in a position which prevents interactions with CO2 This finding is in line with the fact that we could not determine an apparent KM for CO2 with PCSEN_ΔDH and that
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