Figure 8.

Proposed reaction mechanism of 3βHSD/D. Tyr-159, Lys-163, and Arg-326 are proposed to be involved in the dehydrogenation and decarboxylation of 4α-carboxysterol with NAD⁺. 4α-Carboxysterol binding is stabilized by Arg-326. Tyr-159 acts as the general base to abstract the proton of the hydroxyl group of steroid, facilitates the hydride transfer to NAD⁺, and acts as the general acid to deliver a proton to the 3-keto group, and together with Arg-326 it facilitates the decarboxylation of the carboxysteroid. Tyr-159 and a water molecule probably help in the final tautomerization step.