Acyl-CoA Dehydrogenases (ACADs) are a large family of flavoenzymes that catalyze the oxidation of fully saturated acyl-CoA thioesters to their α,β-unsaturated counterparts. Although most notably involved in fatty acid β-oxidation processes across many domains of life, ACADs are also utilized in secondary metabolite biosynthesis in certain bacteria. The work presented here involves the characterization of a bacterial ACAD, TcsD, involved in the biosynthesis of the clinically-relevant <i>Streptomyces</i> polyketide metabolite FK506 (tacrolimus). Specifically, TcsD catalyzes the formation of the γ,δ-olefin of the extender unit allylmalonyl-CoA which is incorporated into the FK506 polyketide scaffold at the C21 position. This unusual transformation cannot be rationalized to proceed via a canonical ACAD mechanism wherein the initial proton abstraction relies of the relatively low pK<sub>a</sub> of the α-carbon of the thioester substrate. Herein, we present biochemical data from experiments using natural substrates and substrate analogs to probe the mechanism of TcsD. The oxidation catalyzed by TcsD is shown to be regiospecific, acting exclusively at the γ,δ-position of fatty acyl-CoA substrates. In addition, we report high resolution (1.75 Angstroms) structural data from the first solved crystal structure of this enzyme. We use this combination of biochemical and structural data to propose a logical chemical mechanism for this novel ACAD activity.

Investigating the Regiospecificity of an Unusual Bacterial Acyl-CoA Dehydrogenase

253rd National Meeting (2017)

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