Catalytic
specificity for the KstR1-regulated acyl-CoA dehydrogenases
and product regio- and stereochemistry for ChsE4-ChsE5. (a) Plot of
the catalytic specificity (kcat/KM) of ChsE4-ChsE5, ChsE3, and ChsE1-ChsE2 for
the three acyl-CoA metabolic intermediates of cholesterol side chain
β-oxidation, 3-OPC-CoA, 3-OCO-CoA, and 3-OCS-CoA, respectively.
(b) ChsE4-ChsE5 forms (24E)-3-oxo-cholest-4,24-dien-26-oyl-CoA
as determined by the 1H NMR spectra (700 MHz) of substrate
precursor 3-oxo-cholest-4-en-26-oic acid and the ChE4-ChsE5 assay
product after alkaline hydrolysis of its thioester, top and bottom,
respectively. The spectra illustrate the changes in the alkene (I)
and methyl (II) regions. The biochemical reaction catalyzed by ChsE4-ChsE5
is shown in Scheme 1.