Decarbonization of the economy will require increased utilization of one-carbon chemicals such as carbon dioxide (CO2) and methane (CH4). Acetylenecarboxylic acid (ACA) can be synthesized from methane and carbon dioxide. Our goal is to transform ACA into value-added chemicals such as malonic acid and 3-hydroxypropionic acid. Here we investigate two tautomerase enzymes, cis-CaaD and Cg10062, in the utilization of ACA for the production of malonate semialdehyde (MSA), a common intermediate of malonic acid and 3-hydroxypropionic acid synthesis. These enzymes have 53% similarity, identical active site amino acid residues, a homologous β-α-β structure, a catalytic proline at the N-terminal, and require no cofactor or metal for catalysis. cis-CaaD catalyzes the formation of MSA as the nearly exclusive product (Km 1160 μM), while Cg10062 affords acetaldehyde (ACH, 81% product formation) as the major product with low amounts of MSA (19% product formation, Km 66 μM). In our ongoing efforts to improve the rate of formation while preserving exclusive MSA production, we have carried out site-specific mutagenesis and protein crystallography to understand the subtle differences between these two enzymes. Here, we present the kinetic characterization of multiple variants of both cis-CaaD and Cg10062 as well as their substrate-soaked atomic resolution crystal structures that may give insight into both the mechanism of reaction and the relative products formed for each variant.

Mechanistic evaluation of tautomerases-catalyzed formation of malonate semialdehyde from acetylenecarboxylic acid

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