Elucidating Structural and Activity Differences Between Tautomerases cis-CaaD and Cg10062: Implications For 3-Hydroxypropionic Acid Production from Acetylenecarboxylic Acid

In efforts to reduce harmful greenhouse gasses such as methane (CH4) and carbon dioxide (CO2), we look to acetylenecarboxylic acid (ACA). ACA may be derived from CH4 and CO2 and converted into high priority target chemicals, such as 3-hydroxypropionic acid which is being targeted to be produced through biocatalytic methods over petroleum-derived, which are non-renewable, produces environmental pollution, and non-sustainable. Here we evaluate the tautomerase enzyme cis-CaaD and its almost exclusive conversion of ACA to intermediate product malonate semialdehyde (MSA) which can further be enzymatically converted to high priority target chemicals. We also compare cis-CaaD to the previously evaluated tautomerase Cg10062 which has 54% similarity yet in its native form does not afford primarily MSA (15% product formation) but rather follows a decarboxylation pathway to produce acetaldehyde (ACH, 85% product formation). Although cis-CaaD produces predominantly MSA, Cg10062 is more catalytically efficient. Through trapping of intermediates in atomic resolution crystal structures we are able to gain a better understanding of the covalent mechanism of cis- CaaD. Structural evaluation of cis-CaaD and Cg10062 variants gives rationale for different product profiles. By evaluating atomic interaction between active site residues, subtle positional changes of residue 114 in Cg10062 versus cis-CaaD lead to either hydration or hydration and decarboxylation. Additionally, structural evaluations of cis-CaaD and Cg10062 chimeras illustrate the C- terminal's necessity in activity by coordination of Arg70 into the active site. This further understanding of cis-CaaD and Cg10062 will enable better bioconversion of ACA to high value chemicals.