Acyl-CoA dehydrogenases (ACADs) are a class ofenzymes that function to catalyze the initial step in each cycle of fatty acidβ-oxidation in themitochondria ofcells. Their action results in the introduction of atransdouble-bond between C2 (α) and C3 (β) of the acyl-CoAthioester substrate.[1]Flavin adenine dinucleotide (FAD) is a required co-factor in addition to the presence of an active siteglutamate in order for the enzyme to function.
The following reaction is theoxidation of thefatty acid by FAD to afford an α,β-unsaturated fatty acid thioester ofcoenzyme A:
ACADs can be categorized into three distinct groups based on their specificity for short-, medium-, or long-chainfatty acidacyl-CoA substrates. While different dehydrogenases targetfatty acids of varying chain length, all types of ACADs are mechanistically similar. Differences in the enzyme occur based on the location of the active site along theamino acid sequence.[2]
ACADs are an important class of enzymes inmammaliancells because of their role in metabolizingfatty acids present in ingested food materials. This enzyme's action represents the first step infatty acid metabolism (the process of breaking long chains of fatty acids into acetyl-CoA molecules). Deficiencies in these enzymes are linked togenetic disorders involvingfatty acidoxidation (i.e. metabolic disorders).[3]
ACAD enzymes have been identified in animals (of which there are 9 major eukaryotic classes), as well as plants,[4] nematodes,[5] fungi,[6] and bacteria.[7] Five of these nine classes are involved in fatty acid β-oxidation (SCAD, MCAD, LCAD, VLCAD, and VLCAD2), and the other four are involved in branched chain amino acid metabolism (i3VD, i2VD, GD, and iBD). Most acyl-CoA dehydrogenases are α4homotetramers, and in two cases (for very long chain fatty acid substrates) they are α2homodimers. An additional class of acyl-CoA dehydrogenase was discovered that catalyzes α,β-unsaturation reactions with steroid-CoA thioesters in certain types of bacteria.[8][9] This class of ACAD was demonstrated to form α2β2 heterotetramers, rather than the usual α4 homotetramer, a protein architecture that evolved in order to accommodate a much larger steroid-CoA substrate.[10][11]
ACADs are classified asEC1.3.99.3.
The medium chain acyl-CoA dehydrogenase (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.[1] This protein is a homotetramer with each subunit containing roughly 400amino acids and one equivalent ofFAD per monomer. The tetramer is classified as a "dimer of dimers" with an overall diameter of approximately 90Å.[2]
The interface between the two monomers of a single dimer of an ACAD contains the FADbinding 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 anacyl-CoA substrate binding site. This gives a total of four FAD molecules and fouracyl-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 overallenzyme stability. Theacyl-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.[1]
MCAD can bind to a rather broad range of chain-lengths in theacyl-CoA substrate, however studies show that its specificity tends to targetoctanoyl-CoA (C8-CoA).[12]
A novel ACAD enzyme architecture in some species of steroid-utilizing bacteria (Actinomycetota andPseudomonadota) was discovered, and is involved in the utilization of ubiquitous steroid substrates like cholesterol by pathogenic organisms likeMycobacterium 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 causedgene 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.
The acyl-CoA dehydrogenase mechanism proceeds through anE2 elimination. This elimination is initiated by aglutamate residue, which, while necessary for the mechanism, is not conserved.[1]
The residue appears in a wide range of locations within the different types of the enzyme (it is Glu 376 in MCAD). The glutamate residue deprotonates thepro-R hydrogen of the alphacarbon.Hydrogen bonding of the substrate's carbonyl oxygen to both the 2'-OH of the ribityl side-chain ofFAD and to the main chain N-H of the previously mentioned glutamate residue lowers thepKa of thisproton, allowing it to be readily removed by glutamate.[1]
As the alphacarbon is being deprotonated, the pro-R hydrogen of the beta carbon leaves as a hydride to FAD in a concerted step. It adds to theRe face of FAD at the N-5 position, and the enzyme holds FAD in place through hydrogen bonding with thepyrimidine portion andhydrophobic interactions with the dimethylbenzene portion. Thesubstrate has now been transformed into an α,βunsaturatedthioester.[1]
As FAD picks up the hydride, the carbonyloxygen adjacent to the N-1nitrogen becomes negatively charged. Theseelectrons are in resonance with the N-1 nitrogen, distributing and stabilizing the resulting negative charge. The charge is also stabilized by hydrogen bonding between the oxygen and nitrogen of interest and various residues within the enzyme.[1]
Deficiencies in acyl-CoA dehydrogenases result in decreased ability to oxidize fatty acids, thereby signifying metabolic dysfunction. Medium-chain acyl-CoA dehydrogenase deficiencies (MCADD) are well known and characterized because they occur most commonly among acyl-CoA dehydrogenases, leading tofatty acidoxidation disorders and the potential of life-threatening metabolicdiseases. Some symptoms of medium-chain acyl-CoA dehydrogenase deficiency include intolerance tofasting,hypoglycemia, andsudden infant death syndrome. These symptoms are seen as directly connected to the inability to metabolizefats. Intolerance tofasting andhypoglycemia result from the inability to gainenergy and makesugar from fat stores, which is how most of humans' excess energy is stored. Also, fatty acids can begin to accumulate in theblood, lowering the blood'spH and causingacidosis.[1]
MCAD is related to / has an association withsudden infant death. Approximately 90% of cases of MCAD are due to a single pointmutation where thelysine at position 304 (Lys304) is replaced by aglutamate residue and this prevents the enzyme from properly functioning.[1] It is reported that, every year, 1 in 20,000infants is born with adeficiency in his/her medium-chain acyl-CoA dehydrogenases that is caused by a mutation. The mutation isrecessive, and oftenparents ofchildren who have the deficiency can be diagnosed afterward as carriers.[3]
Inhumans the most common naturally occurring mutation in MCAD is located atamino acid residue Lys-304.[1] The altered residue occurs as a result of a single-point mutation in which the lysine side chain is replaced by a glutamate. Lys-304 typically interacts with surrounding amino acid residues by forminghydrogen bonds with Gln-342, Asp-300, and Asp-346. When a mutation causes glutamate to take the place of lysine, an additional negative charge is introduced at that site, which disrupts the normally occurring H-bonding. Such a disruption alters the folding pattern of the enzyme, ultimately compromising its stability and inhibiting its function in fatty acid oxidation.[12] The efficiency of the mutatedprotein is about 10 times lower than that of the natural protein.[13] This can lead to the symptoms of the deficiency listed above.
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