Enzymatic Conversion of CO2: From Natural to Artificial Utilization

Abstract

Enzymatic carbon dioxide fixation is one of the most important metabolic reactions as it allows the capture of inorganic carbon from the atmosphere and its conversion into organic biomass. However, due to the often unfavorable thermodynamics and the difficulties associated with the utilization of CO₂, a gaseous substrate that is found in comparatively low concentrations in the atmosphere, such reactions remain challenging for biotechnological applications. Nature has tackled these problems by evolution of dedicated CO₂-fixing enzymes, i.e., carboxylases, and embedding them in complex metabolic pathways. Biotechnology employs such carboxylating and decarboxylating enzymes for the carboxylation of aromatic and aliphatic substrates either by embedding them into more complex reaction cascades or by shifting the reaction equilibrium via reaction engineering. This review aims to provide an overview of natural CO₂-fixing enzymes and their mechanistic similarities. We also discuss biocatalytic applications of carboxylases and decarboxylases for the synthesis of valuable products and provide a separate summary of strategies to improve the efficiency of such processes. We briefly summarize natural CO₂ fixation pathways, provide a roadmap for the design and implementation of artificial carbon fixation pathways, and highlight examples of biocatalytic cascades involving carboxylases. Additionally, we suggest that biochemical utilization of reduced CO₂ derivates, such as formate or methanol, represents a suitable alternative to direct use of CO₂ and provide several examples. Our discussion closes with a techno-economic perspective on enzymatic CO₂ fixation and its potential to reduce CO₂ emissions.

Scheme 1. Standard Molar Gibbs Energy of Formation (ΔfG°) and Experimental Reduction Potentials of Carbon Dioxide, Carbon Monoxide, Formic Acid, Formaldehyde, Methanol, and Methane, at 298.15 K in kJ/mol (Oxidation States of Carbon Are Given As Roman Numerals)^,

Scheme 2. General Mechanistic Steps of Enzymatic Carboxylations

Scheme 3. Mechanistic Strategies for the Activation of the Nucleophile (Enol- or Enamine-Formation) and CO₂

(I) Endiolate stabilized by Mg²⁺, formed in the mechanism of RuBisCO. (II) Enolate formed by acetyl-CoA carboxylases. (III) Phenolate bound to Zn²⁺ in the active site of bivalent metal-dependent decarboxylases. (IV) Trienolate attack onto CO₂ by the cofactor-independent decarboxylases. (V) Hydride attack (NADPH) onto crotonyl-CoA by enoyl-CoA carboxylases/reductases, forming the intermediary enolate. (VI) Cleavage of the phosphate ester of phosphoenolpyruvate by pyruvate carboxylase. (VII) Carboxyphosphate, formed either from phosphoenolpyruvate or ATP and bicarbonate. (VIII) Formation of carboxybiotin by acetyl-CoA carboxylases. (IX) Intermediary dienolate, covalently bound to prFMN, in the mechanism of AroY-type enzymes. (X) Umpolung of the carbonyl center of either acyl-CoA (2-ketoglutarate synthase or pyruvate synthase) or aldehyde substrates (pyruvate decarboxylase) with TPP as cofactor.

Scheme 4. Nature’s Strategies for Reducing the Free Energy of Carboxylation Reactions

Scheme 5. Catalytic Cycle of RuBisCO; Changes of the Oxidation State of Selected Carbons Are Given in Roman Numerals

Figure 1

Active site of Spinacia oleracea RuBisCO (PDB 8RUC) lies at the dimer–dimer interface of two large subunits (pink and green) and features a Mg²⁺ ion (green). Here, the active site is occupied by the transition state analogue 2-carboxyarabinitol bisphosphate (CABP, forest green).

Figure 2

Active site of E. coli PEPC (PDB 1JQN) with bound Mn²⁺ (instead of naturally occurring Mg²⁺) and the substrate analogue 3,3-dichloro-2-phophonomethyl-acrylic acid (DCO) bound in the active site.

Scheme 6. Catalytic Cycle of PEPC; Changes of the Oxidation State of Selected Carbons Are Given in Roman Numerals

Scheme 7. Catalytic Cycle of Acetyl-CoA/Propionyl-CoA Carboxylases

Changes of the oxidation state of selected carbons are given in roman numerals. BC, biotin carboxylase; BCCP, biotin carboxyl carrier protein; CT, carboxytransferase.

Scheme 8. Catalytic Mechanism of Oxoacid:Ferredoxin Oxidoreductases; Oxidation States of Selected Carbons Are Highlighted in Roman Numerals

Figure 3

Active site of 2KFOR from Magnetococcus marinus with a TPP-bound succinyl-CoA intermediate present (PDB 6N2O). The carbon atoms of succinate are highlighted in pink.

Scheme 9. Catalytic Cycle of Isocitrate Dehydrogenase When Run in Reductive TCA Cycle; Changes of the Oxidation State of Selected Carbons Are Given in Roman Numerals

Figure 4

Crystal structure of the H-protein of the glycine cleavage system of Pisum sativum (PDB 1HPC) with the lipoic acid anchored to a lysine (highlighted in orange).

Scheme 10. Mechanism of the Glycine Cleavage System; Changes of the Oxidation State of Selected Carbons Are Given in Roman Numerals.

Figure 5

Molybdenum-dependent formate dehydrogenase (FDH). (A) Proposed catalytic cycle of molybdenum-dependent FDHs. (B) Active site of the reduced molybdenum-dependent FDH from Escherichia coli (PDB 1AA6).

Figure 6

Active site of the [NiFe] CO dehydrogenase from Carboxydothermus hydrogenoformans with bound CO₂ (PDB 3B52).

Scheme 11. Catalytic Cycle of [NiFe] CO Dehydrogenases

Scheme 12. Catalytic Cycle of Crotonyl-CoA Carboxylation by Crotonyl-CoA Carboxylase/Reductase

Figure 7

Active site of Kitasatospora setae crotonyl-CoA carboxylase/reductase (PDB 6OWE) with ethylmalonyl-CoA, the product of the carboxylation reaction, and NADP⁺ bound in the active site. Key active site residues necessary for accommodation of CO₂ (His, Asn, Phe, and Glu) are highlighted in orange.

Scheme 13. Enzymatic ortho-Carboxylation of (A) Resorcinol, (B) Phenol, (C) Catechol, and (D) meta-Aminophenol by Different Bivalent Metal-Dependent Decarboxylases^,,,,,

The displayed numbers correspond to conversions to the respective product. n.d. = not determined or not found.

Figure 8

(A) View of the active site of the 2,3-DHBD_Fo from Fusarium oxysporum, bound substrate catechol (yellow). The catalytic Zn²⁺ is displayed as a green ball, and the complexing amino acids (Glu8, His167, and Asp291) and the catalytic triad (Asp291, His222, and Glu225) are displayed as sticks (PDP 7BP1). (B) General mechanism of the (de)carboxylation of phenol by a bivalent metal-dependent decarboxylase (Zn²⁺ is shown as example and can be Mg²⁺ or Mn²⁺ in other enzymes).

Figure 9

Active site of the phenolic acid decarboxylase from Bacillus subtilis. Amino acid residues involved in the hydrogen-bonding network of the substrate (Tyr11, Tyr13) and the carboxylating source (Tyr19, Tyr66) as well as the glutamate residue acting as the catalytically important general acid are displayed as sticks (PDP 2P8G).

Scheme 14. Catalytic Cycle of the Carboxylation, Catalyzed by Phenolic Acid Decarboxylases, Shown for para-Vinylphenol by a Cofactor-Independent Phenolic Acid Decarboxylase^,

Figure 10

General substrate scope of the side chain carboxylation of styrene derivatives by phenolic acid decarboxylases.

Scheme 15. Overview on prFMN-Dependent Decarboxylation Reactions: (A) Decarboxylation of Cinnamic Acids by Ferulic Acid Decarboxylases; (B) Decarboxylation of Phenolic Substrates by AroY Type Enzymes; (C) Decarboxylation of Heteroaromatic Substrates by Pyrrole-2-carboxylate Decarboxylase from Pseudomonas aeruginosa

Scheme 16. Biosynthesis of the prFMN Cofactor (prFMNH₂) by the UbiX Enzyme

Oxidative maturation of the bound cofactor is required to reach the catalytically active iminium form (prFMNH^iminium). Free prFMNH₂ in the presence of oxygen degrades to prFMN-hydroperoxide (prFMNH C4a-OOH).

Figure 11

Crystal structure of the active site of Fdc1 from A. niger (PDB 4ZAB) with α-fluoro cinnamic acid (yellow). The substrate is positioned with its α-carbon near prFMN C1′ and with its β-carbon on top of prFMN C4a. The residues Arg173, Glu277, and Glu282 (orange) constitute the catalytic triad.

Scheme 17. Proposed Reaction Mechanism for Ferulic Acid Decarboxylases Decarboxylating Cinnamic Acid-Type Substrates Based on a Dipolar 1,3-Cycloaddition

Scheme 18. Proposed Reaction Mechanism for Decarboxylation of Phenolic Substrates by AroY-Type Enzymes

Figure 12

Crystal structure of EcAroY reconstituted with prFMN without substrate (PDB 5O3N). The amino acids marked in orange (Arg171, Glu289) contribute with hydrogen bonding to the substrate carboxylate moiety, and residues colored in yellow (Arg188, His327, Lys363, His436) are in hydrogen bonding proximity to the hydroxy group of the substrate according to DFT calculations.

Scheme 19. Proposed Electrophilic Aromatic Substitution for Decarboxylation via HudA-Type Enzymes

Figure 13

Crystal structure of pyrrole-2-carboxylic acid decarboxylase HudA from Pseudomonas aeruginosa with the reversible inhibitor imidazole bound in a covalent prFMN-imidazole adduct (PDB 7ABN). A key role is assigned to Glu278 and Asn318 in the decarboxylation of heteroaromatic compounds.

Scheme 20. Substrates That Were Accepted for Carboxylation by prFMN-Dependent Enzymes

Red arrows indicate the position of carboxylation.^,,,,,,,, For each substrate the carboxylating enzyme, the carboxylating agent and the results in terms of carboxylation activity indicated by conversion are shown (see also Table 4).

Scheme 21. Application of TPP-Dependent Decarboxylases for Carboxylations

(A) Two-step enzymatic cascade towards the synthesis of L-Met starting from methional via a carboxylation and subsequent amination step. (B) Catalytic cycle of the decarboxylation of keto acids by TPP-dependent decarboxylases.

Scheme 22. Decarboxylation of Gallic Acid and Protocatechuic Acid by the Two Fungal Enzymes AGDC1 and PPP2

Scheme 23. Reaction Engineering of Biocatalytic Carboxylation Processes

Figure 14

Natural carbon fixation cycles. (A) CBB cycle, (B) rTCA and roTCA cycle, (C) acetogenic WL pathway (turquoise) and reductive glycine pathway. (D) dicarboxylate cycle (orange). (E) 3HP/4HB cycle (blue) and 3HP bicycle (magenta). Carboxylation steps are highlighted.

Figure 15

Design and realization of synthetic CO₂ fixation pathways. (A) The theoretical considerations for the creation of new-to-nature pathways are (1) selection of a suitable carboxylase, (2) design of a pathway around the chosen carboxylase, and (3) pathway evaluation. (B) The experimental workflow for the assessment and optimization of new-to-nature (carboxylation) pathways entails (1) characterization of single enzymes, (2) reconstruction of pathway modules, and (3) full in vitro pathway assembly and optimization.

Scheme 24. Reductive THF Cascade

The carbon of formate is reduced in a stepwise fashion from oxidative state +II to −II. The oxidative state of the relevant carbon atom is indicated in roman numerals. FTS = formyl-THF synthase, MCH = methenyl-THF cyclohydrolase, MTD = methylene-THF dehydrogenase, MTR = methylene-THF reductase.

Scheme 25. Reaction Mechanism at the NiFeS Cluster of ACS

Note that the shown mechanism represents only one possibility. Alternative mechanisms involving Ni(0) species have also been proposed..

Scheme 26. Reaction Mechanism of SHMT

Mechanism for the hydrolysis of 5,10-methylene tetrahydrofolate to formaldehyde and THF followed by the aldol condensation of glycine with formaldehyde to serine. The oxidative state of the relevant carbon atoms is indicated in roman numerals.

Scheme 27. Reaction Mechanism of HPS Showing Ru5P Condensation with Formaldehyde; The Oxidation States of Relevant Carbon Atoms Are Given in Roman Numeral

Figure 16

(A) Serine cycle and (B) homoserine cycle. Carboxylation/C₁ elongation steps are highlighted.

Figure 17

Crystal structure of G. stearothermophilus SHMT (PDB 1KL2(349)) with 5-formyl-tetrahyrofolate (5-fTHF; orange) and glycyl-PLP adduct (yellow). The two subunits that create the active site are indicated in different colors. The postulated general bases glutamate (green) and histidine (blue) are shown as sticks.

Figure 18

Crystal structure of M. gastri HPS (PDB 3AJX) with ribulose-5-phosphate (Ru5P) modeled in from E. coli 3-keto-l-gulonate-6-phosphate decarboxylase structure (PDB 1XBV). The Mg²⁺-coordinating charged residues, as well as the histidine base, are shown as sticks.

Scheme 28. Postulated TPP-Dependent Mechanism of DAS; The Oxidation State of Relevant Carbon Atoms Are Given in Roman Numerals

Figure 19

Crystal structure of E. coli PFL (PDB 3PFL). The relevant active site residues are shown as sticks. Oxamate (beige) is used as a substrate analogue in place of the natural substrate pyruvate.

Scheme 29. Radical Mechanism for the Condensation of Formate and Acetyl-CoA Forming Pyruvate, Catalyzed by PFL; The Oxidation State of Relevant Carbon Atoms Are Given in Roman Numerals

Scheme 30. Industrial CO₂ Production

(A) CO₂ is a byproduct of hydrogen production by steam reforming. The CO₂ is removed from the product mixture by absorption in an aqueous solution of (B) potassium carbonate or (C) ethanolamine at high pressure and low temperature, and subsequently released by raising the temperature and lowering the pressure.

Scheme 31. Fermentative Production of Succinic Acid from Glycerol and CO₂

(A) Overall reaction equation. (B) Metabolic pathway of Mannheimia succiniciproducens. Glycerol is converted to phosphoenolpyruvate, PEP, via dihydroxyacetone. PEP, carboxykinase; PEPCK, carboxylates PEP to oxaloacetate and transfers the phosphate group to ADP to produce ATP. Malate dehydrogenase, MDH, reduces the α-keto-acid to the α-hydroxy-acid malate, which is dehydrated by fumarate hydratase, FH, to fumarate. Fumarate reductase, FRD, reduces fumarate to succinate, using quinol as hydrogen donor.