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Abstract:
<p>2-Hydroxyacyl-CoA lyase/synthase (HACL/S) is a thiamine diphosphate (ThDP)-dependent versatile enzyme originally discovered in the mammalian α-oxidation pathway. HACL/S natively cleaves 2-hydroxyacyl-CoAs and, in its reverse direction, condenses formyl-CoA with aldehydes or ketones. The one-carbon elongation biochemistry based on HACL/S has enabled the use of molecules derived from greenhouse gases as biomanufacturing feedstocks. We investigated several HACL/S family members with high activity in the condensation of formyl-CoA and aldehydes, and distinct chain-length specificities and kinetic parameters. Our analysis revealed the structures of enzymes in complex with acyl-CoA substrates and products, several covalent transition states, bound ThDP and ADP, as well as a previously unseen C-terminal active site region. One of these new states corresponds to the intermediary α-carbanion with ThDP covalently attached to formyl- CoA. This research distinguishes HACL/S from related sub-families and identifies key residues involved in substrate binding and catalysis. These findings expand our knowledge of acyloincondensation biochemistry and offer new prospects for biocatalysis using carbon elongation.</p>
