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

Abstract

The biologically important, FAD-containing acyl-coenzyme A (CoA) dehydrogenases (ACAD) usually catalyze the anti-1,2elimination 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 highresolution 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.

Ethoxylate Polymer-Based 96-Well Screen for Protein Crystallization

“…Cyclohex-1ene-1-carboxyl-CoA dehydrogenases (CH1enDH) play a role in fermentative cyclohexane carboxylic acid degradation. The homotetrameric enzyme (Mr 164 kDa) from Geobacter metallireducens (growth temperature of 30 • C) was heterologously produced in E. coli with an N-terminal His-tag [19]. Benzoyl-CoA reductase I is involved in the aromatic degrada-tion pathway.…”