Structure of the medium-chain<unk>  tetramer. FAD molecules are shown in yellow. PDB code: 1egc. The medium chain<unk>  (MCAD) is the best known structure of all ACADs, and is the most commonly deficient enzyme within the class that leads to metabolic disorders in animals. This protein is a homotetramer with each subunit containing roughly 400 amino acids and one equivalent of FAD per monomer. The tetramer is classified as a “dimer of dimers” with an overall diameter of approximately 90 Å. The interface between the two monomers of a single dimer of an ACAD contains the FAD binding sites and has extensive bonding interactions. In contrast, the interface between the two dimers has fewer interactions. There are a total of 4 active sites within the tetramer, each of which contains a single FAD molecule and an<unk> -CoA substrate binding site. This gives a total of four FAD molecules and four<unk> -CoA substrate binding sites per enzyme. FAD is bound between the three domains of the monomer, where only the nucleotide portion is accessible. FAD binding contributes significantly to overall enzyme stability. The<unk> -CoA substrate is bound completely within each monomer of the enzyme. The active site is lined with the residues F252, T255, V259, T96, T99, A100, L103, Y375, Y375, and E376. The area of interest within the substrate becomes wedged between Glu 376 and FAD, lining up the molecules into an ideal position for the reaction. MCAD can bind to a rather broad range of chain-lengths in the<unk> -CoA substrate, however studies show that its specificity tends to target octanoyl-CoA (C8-CoA). A novel ACAD enzyme architecture in some species of steroid-utilizing bacteria (Actinomycetota and Pseudomonadota) was discovered, and is involved in the utilization of ubiquitous steroid substrates like cholesterol by pathogenic organisms like Mycobacterium tuberculosis. Genetically, the structure is encoded by two separate genes (open reading frames) that form an obligate α2β2 heterotetramic complex. The structure was most likely the result of an evolutionary event that caused gene duplication and partial loss of function, since half of the FAD cofactor binding residues are in each gene, and only make a complete binding site when expressed together as a complex. This probably allowed for the substrate binding site to open up considerably to accommodate much larger polycyclic-CoA substrates, rather than fatty acids of varying chain lengths.
Acyl-CoA dehydrogenase
Pyruvate carboxylase
Phenylalanine hydroxylase
Aldehyde dehydrogenase
Glutamate dehydrogenase
Butyrate esterase
Glycogen phosphorylase isoenzyme BB
Monoamine oxidase B