Awakening the Sleeping Carboxylase Function of Enzymes: Engineering the Natural CO2-Binding Potential of Reductases

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.

T o harvest atmospheric CO 2 as a sustainable carbon source for (bio)catalytic and (bio)technological applications, 1−5 it is necessary to extend the repertoire of CO 2 -fixing reactions. One possibility is to engineer a carboxylation function into the scaffold of non-CO 2 -fixing enzymes. Generally, the interaction of CO 2 with proteins is poorly understood. 6 However, for enoyl-CoA carboxylase/reductase from Kitasatospora setae (ECR Ks ), four conserved amino acids that form a CO 2 -binding pocket at the active site were described recently 7 (Figure 1a). These four amino acids anchor and position the CO 2 molecule during catalysis, in which a reactive enolate is formed that attacks the CO 2 . 8 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 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). The PCS family clusters closely to ECRs and shows a fully conserved CO 2 -binding motif across individual family members ( Figure S1). 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). We decided to test selected members of these enzyme families in their CO 2 -fixing capabilities.
PCS is a three-domain fusion enzyme that catalyzes the overall conversion of 3-hydroxypropionate to propionyl-CoA 10 ( Figure 2a). The enzyme forms a central reaction chamber, in which three subsequent reactions take place in a synchronized fashion. 11 When we assayed PCS from Erythrobacter sp. NAP1, PCS EN , at 4.4 mM dissolved CO 2 , we detected minor amounts of methylmalonyl-CoA besides the main product propionyl-CoA. Incorporation of 13 CO 2 -label confirmed the latent carboxylation activity of PCS EN (Figure 2b). Notably, the carboxylation function was not limited to the Erythrobacter enzyme, but was also detected with PCS from Chloroflexus aurantiacus (PCS Ca , Table S1).   7 The CO 2 -binding pocket is defined by four conserved residues (Asn81, Phe170, Glu171, His365). CO 2 was modeled into the structure. (b) Maximumlikelihood tree of the MDR superfamily 9 with (potential) CO 2binding enzyme families highlighted in color. The last reaction in the three-reaction sequence of PCS is the reduction of acrylyl-CoA to propionyl-CoA, catalyzed by a reductase domain harboring the CO 2 -binding motif ( Figure  2a,c). We directly tested the reductase domain for carboxylation activity with an E1027Q variant of PCS EN (PCS EN_ΔDH ) that is unable to generate acrylyl-CoA. When PCS EN_ΔDH was provided with external acrylyl-CoA and 4.4 mM dissolved CO 2 , 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.
To improve further the carboxylation efficiency of PCS EN , we compared the active site of PCS EN (PDB: 6EQO) with ECR Ks . While the NADPH binding site, as well as the four CO 2 -binding pocket residues are structurally conserved (Figure 2c), we noticed differences in the second shell of the active site. ECR Ks features a small hydrophilic residue (Thr82), which interacts with Asn81 that stabilizes CO 2 through its carboxyamide NH 2 group. The corresponding residue in PCS EN is occupied by an aspartate (Asp1302). Molecular dynamics (MD) simulations demonstrated that Asp1302 in PCS EN forms a strong anionic hydrogen bond to the carboxamide NH 2 group of Asn1301 (Figures 3a and S5), locking Asn1301 in a position which prevents interactions with CO 2 . This finding is in line with the fact that we could not determine an apparent K M for CO 2 with PCS EN_ΔDH and that  11 ) and ECR Ks (blue 7 ), both cocrystallized with NADP + . Acrylyl-CoA and CO 2 are modeled into the active site. WebLogo-Illustration 12,13 of conserved active site residues using 129 PCS and 29 ECR sequences. Numbering according to PCS EN or ECR Ks , respectively.

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Communication replacing Asn1301 by an aspartate abolished carboxylation activity.
We aimed at unlocking Asn1301 from its fixed position by replacing Asp1302 with different small hydrophilic residues. PCS EN_ΔDH variant D1302S (Figure 3b) showed an increased carboxylation yield of 20.9 ± 0.7% at 4.4 mM dissolved CO 2 , and notably also Michaelis−Menten-like behavior with CO 2 at an apparent K M_CO2 of 27 ± 5 mM (Table 1). Together with MD simulations that showed a more flexible asparagine residue ( Figure S5) this demonstrated that unlocking Asn1301 improves CO 2 -binding and carboxylation efficiency in PCS EN .
Another, equally important catalytic principle in carboxylases is the exclusion of water from the active site to minimize protonation reactions which would prematurely quench C−C bond formation. 7,14−16 In ECR Ks , a conserved methionine (Met356) restricts access of water to the CO 2 -binding pocket. In PCS EN , this residue is a threonine, which presumably allows water to enter the active site and displace the CO 2 molecule (Figure 3c). When we introduced the methionine in PCS EN (PCS EN_ΔDH T1753M, Figure 3d), carboxylation yield increased to 10 ± 2% at 4.4 mM dissolved CO 2 . When combining the D1302S with the T1573M mutation, the carboxylation yield of PCS EN_ΔDH further increased up to 69 ± 3% at 4.4 mM CO 2 ( Table 1). Under saturating CO 2 concentrations (i.e., 44 mM CO 2 ), PCS EN_ΔDH D1302S T1753M showed a carboxylation yield of 94.5 ± 0.7%, demonstrating that we successfully converted the reductase domain into a carboxylase. During engineering, the k cat. of reduction was strongly decreased, while the apparent k cat. for carboxylation was maintained (Table S2) and falls in the range of naturally existing ECRs. 4,17 The engineered carboxylase domain also improved carboxylation yield in the context of the overall reaction of PCS EN (Supporting Information I).
We next investigated the carboxylation potential in the AER enzyme family of unknown function. We chose Nmar_1565 (AER Nm ), a homologue from Nitrosopumilus maritimus, in which two of the four amino acids of the CO 2 -binding motif, namely Phe122 and Glu123, are conserved (Figure 4a,b). Although no function was assigned to AER Nm so far, we speculated that the enzyme might catalyze the reduction of acrylyl-CoA in the 3-hydroxypropionate/4-hyroxybutyrate cycle of N. maritimus. 18,19 Indeed, the enzyme reduced acrylyl-CoA to propionyl-CoA at an apparent k cat. of 0.99 ± 0.11 s −1 , confirming its reductase function.
AER Nm activity was very sensitive to salt and buffer composition (Supporting Information II). When we incubated the enzyme with NaHCO 3 , at concentrations corresponding to 1.31 mM free CO 2 , activity dropped 10-fold. However, under these conditions AER Nm showed a latent carboxylation activity and converted acrylyl-CoA into methylmalonyl-CoA at a carboxylation yield of 4.5 ± 0.9% (Table 2), despite the lack of two of the four amino acid residues of the CO 2 -binding motif. To increase the carboxylation efficiency of AER Nm we decided to rebuild the CO 2 -binding pocket through introduction of asparagine and histidine. Reintroduction of histidine failed due to inactive protein, which might be a result of interrupted second-shell interactions to Thr307 or steric clashes. However, replacing Asp50 by asparagine increased carboxylation yield dramatically (to 82 ± 5%, Figure 4c, Table 2). The increase in catalytic activity in AER Nm D50N was accompanied by an improved K M_CO2 (0.18 ± 0.03 mM), indicating increased CO 2 -binding. AER Nm D50N performed best in 100 mM phosphate buffer, where it showed k cat. and carboxylation yields comparable to those of naturally existing carboxylases, such as RubisCO 20 (Table 2).
In conclusion, we successfully reshaped the energy landscape of acrylyl-CoA reductases from the thermodynamically favored product propionyl-CoA (Δ r G′ 0 ≈ −63 kJ/mol) to the disfavored methylmalonyl-CoA (Δ r G′ 0 ≈ −43 kJ/mol). 23 Our engineering efforts show that improving CO 2 -binding (reduced energy barrier for carboxylation) and minimizing side reaction with water (increased energy barrier for reduction) are both required to establish a carboxylation activity in the scaffold of different reductases. This is in line with the idea that in catalysis stabilization of favorable transition states ("positive catalysis") and destabilization of unwanted transition states ("negative catalysis") are both important, 24−26 as further supported by the finding that suppression of competing protonation side reactions is essential for efficient CO 2 -fixation (a) Active sites of AER Nm (salmon) and ECR Ks (blue). Illustration of conserved active site residues, generated by WebLogo 12,13 using 21 AER Nm and 29 ECR sequences. Residue numbering refers to AER Nm or ECR Ks sequence, respectively. (b) Model of the AER Nm wild type active site carrying an Asp50 instead of a conserved Asn. (c) Model of the AER Nm D50N active site. Homology models were created with an ECR from Streptomyces sp. NRRL 2288 (PDB: 4y0k 21 ) using SWISS-MODEL. 22 Acrylyl-CoA and CO 2 were modeled into the active site.

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Communication in ECR Ks and 2-ketopropyl coenzyme M oxidoreductase/ carboxylase. 14−16 On a broader picture, our findings also raise questions about the emergence of natural carboxylases. How did carboxylation functions naturally evolve in the scaffold of proteins, such as RubisCO or ECR? It has been suggested that these enzymes originated from non-CO 2 -fixing ancestors. 27,28 Our data provides experimental evidence for this evolutionary scenario by demonstrating that the MDR superfamily, to which ECR belongs, naturally possesses the capacity to interact with the CO 2 -molecule. It apparently takes only a few mutations to transform latent carboxylases that convert CO 2 at low efficiency and nonphysiological CO 2 concentrations into decent CO 2 -fixing enzymes.
Another apparent question is why PCS and AER would possess a "sleeping carboxylase function"? One explanation might be that the latent carboxylation activity was selected for. PCS operates in the 3-hydroxypropionate bicycle in C. aurantiacus and a modified version thereof in Erythrobacter sp. NAP1 ( Figure S7a), 29,30 while AER Nm presumably works in the 3-hydroxypropionate/4-hyroxybutyrate cycle in N. maritimus ( Figure S7b). 18 Bioenergetic considerations suggest that even a low carboxylation activity would increase biomass yield of these organisms, which thrive at a constantly low energy supply 18 (Supporting Information III).
In summary, our proof-of-principle study demonstrates that it is possible to exploit the active site of reductases to create novel carboxylases. This opens the possibility for the future engineering of novel CO 2 -fixing enzymes that could find application in biocatalysis and synthetic biology (e.g., in artificial pathways for the conversion of CO 2 31,32