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

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Acyl-coenzyme A dehydrogenases (ACADs) are crucial enzymes catalyzing proton and hydride elimination in aliphatic thioesters.
• Their typical reaction involves anti-1,2-elimination, essential for various metabolic pathways.

Purpose of the Study:

• To elucidate the structure and function of an ACAD from anaerobic bacteria with an unusual catalytic mechanism.
• To understand the structural basis for the enzyme's novel reactivity and explore its biocatalytic potential.

Main Methods:

• High-resolution crystal structure determination of cyclohex-1-ene-1-carboxyl-CoA (Ch1CoA) dehydrogenase.
• Biochemical assays to characterize enzyme activity and substrate specificity.
• Structure-guided site-directed mutagenesis to investigate catalytic mechanisms.

Main Results:

• An anaerobic bacterial ACAD was found to catalyze unprecedented 1,4-eliminations of Ch1CoA to cyclohex-1,5-diene-1-carboxyl-CoA (Ch1,5CoA) and subsequently to benzoyl-CoA.
• Structural analysis revealed a catalytic aspartate at C3 and the absence of a catalytic glutamate at C1, explaining the unorthodox reactivity.
• Enzyme engineering restored the typical C1,C2-dehydrogenation activity, demonstrating the role of specific amino acid residues.

Conclusions:

• The study provides a structural rationale for the extended catalytic capabilities of ACADs beyond typical anti-1,2-eliminations.
• The findings offer novel biocatalytic routes for synthesizing cyclic 1,3-diene building blocks, expanding the enzyme's synthetic utility.