Structural Basis of Cyclic 1,3‐Diene Forming Acyl‐Coenzyme A Dehydrogenases

The biologically important, FAD‐containing acyl‐coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti‐1,2‐elimination of a proton and a hydride of aliphatic CoA thioesters. Here, we report on the structure and function of an ACAD from anaerobic bacteria catalyzing the unprecedented 1,4‐elimination at C3 and C6 of cyclohex‐1‐ene‐1‐carboxyl‐CoA (Ch1CoA) to cyclohex‐1,5‐diene‐1‐carboxyl‐CoA (Ch1,5CoA) and at C3 and C4 of the latter to benzoyl‐CoA. Based on high‐resolution Ch1CoA dehydrogenase crystal structures, the unorthodox reactivity is explained by the presence of a catalytic aspartate base (D91) at C3, and by eliminating the catalytic glutamate base at C1. Moreover, C6 of Ch1CoA and C4 of Ch1,5CoA are positioned towards FAD‐N5 to favor the biologically relevant C3,C6‐ over the C3,C4‐dehydrogenation activity. The C1,C2‐dehydrogenation activity was regained by structure‐inspired amino acid exchanges. The results provide the structural rationale for the extended catalytic repertoire of ACADs and offer previously unknown biocatalytic options for the synthesis of cyclic 1,3‐diene building blocks.