The acyl-CoA dehydrogenases (ACADs), CasL-CasN from Rhodococcus jostii RHA1 and Tcur3481-Tcur3483 from Thermomonospora curvata DSM 43183, were heterologously expressed and purified. Tcur3481-Tcur3483 has an 18-fold higher specificity constant for the 3-carbon side chain steroid metabolite compared to the five carbon side chain metabolite. Tcur3481-Tcur3483 structure was determined to a resolution of 2.1 Å. The protein adopted an α22 structure, similar to the structure of the Mycobacterium tuberculosis ACAD FadE26-FadE27. The putative substrate binding site of Tcur3483 is 1.6 times smaller in volume than FadE26 and the catalytic glutamate base is part of a helix while the corresponding residue in FadE26 part of a loop. These differences may contribute to the broader substrate specificity of FadE26. The molecular determinants of substrate specificity were also tested by creating the Ala691Gly variant of the M. tuberculosis ACAD, FadE34. The variant gained the ability to utilize 3-carbon isopropyl side chain metabolite as a substrate.