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.Human very-long-chain acyl-CoA dehydrogenase
Created by Alan Molina
Human very-long-chain acyl-CoA dehydrogenase (VLCAD) ( pdb ID: 3B96) is an enzyme associated with the internal mitochondrial membrane. It has a molecular weight of 63388.02 g/mol and an isoelectric point of 6.91. VLCAD belongs to the ACAD family of enzymes which includes short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and dehydrogenases associated with amino acid metabolism (1). Important structural differences between VLCAD and other ACAD family members exist which underlie functional differences (1,2). The differences between VLCAD and MCAD (PDB ID: 3MDD A) are of particular importance.
VLCAD catalyzes the first of four steps necessary for the beta-oxidation of fatty acids (1). The beta-oxidation of fatty acids is a catabolic pathway crucial to the conversion of the energy stored in fatty acids into a usable form via the production of high-energy molecules such as ATP. Beta-oxidation converts acyl-CoA into acetyl-CoA and produces one molecule of FADH2 and NADH. Acetyl-Coa is the entry molecule for the citric acid cycle. It provides a continuous source of carbons and is also produced by glycolysis (3).
Specifically, VLCAD catalyzes the dehydrogenation of the C2-C3 bond of fatty acyl-CoA chains which are twelve to twenty-four carbons long to produce trans-2,3-enoyl-CoA (1). .
The dehydrogenation reaction follows bimolecular elimination kinetics. The reaction is initiated by a glutamate residue as it removes a proton from C2. The resulting non-bonded electrons create an unstable transition state which is relieved upon transfer of hydride from the C3 to flavin adenine dinucleotide (FAD) - a crucial ligand associated with VLCAD. This results in the formation of a trans double-bond between C2 and C2. The key to the enzymatic activity of VLCAD is its stabilization of the transition state which effectively decreases the activation energy for the reaction (4). Trans-2,3-enoyl-CoA is not released from the enzyme until electron transfer flavoprotein (ETF) reoxidizes FADH. ETF-ubiquinone oxidoreductase transfers the electrons from ETF into the citric cycle reinforcing the relationship between VLCAD and the important respiration cycle in the mitochondria (4).
VLCAD and MCAD have similarities in both their primary and tertiary structures. The E value obtained through the online Basic Local Alignment Search Tool was 3e-34. The Z and rmsd values obtained through the Dali server were 39.0 and 2.1 respectively (5). The differences in primary and tertiary structure correspond to slight differences in function (1,2).
Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a homodimer associated with the inner-mitochondrial membrane. Each monomer is composed of four structural domains with characteristic secondary structures. The four domains from the N-terminal to the C-terminal include an alpha-helical domain composed of six alpha-helices, a beta-sheet domain composed of seven beta-sheets, an alpha-helical domain composed of five helices, and an alpha-helical bundle composed of six alpha helices. Additionally, five separate sequences of random coils, each four to seven residues long, are interspersed through all four domains. The C-terminal alpha-helical bundle is approximately 180 residues long and is not present in other ACAD enzymes. It is positioned perpendicular to the N-terminal helical domains (1). Each monomer binds FAD as a cofactor and an acyl-CoA chain 12-24 carbons long as a substrate, yet dimerization is still necessary for enzymatic function (6).
The catalytic site of VLCAD is a 2.4 nm long cavity bordered by hydrophobic residues between two alpha-helical domains and the beta-sheet domain. The acyl-CoA chain inserts with its C2-C3 atoms between the re-face of FAD and the catalytic Glu-422 residue. The long hydrocarbon tail is stabilized by van der Waals interaction with hydrophobic residues lining the cavity the most important of which are Gly-135, Ile-136, Leu-138, Val-124, Val-127, Phe-421, and Ala-307. These residues are particularly important becuase they play in important role in stabilization of longer acyl-CoA chains. Catalysis occurs when Glu-422 abstracts a proton from C2 of the acyl-CoA chain followed by the transfer of hydride from C3 to the riboflavin moiety of FAD to form a C2-C3 double bond. The active site positions FAD and the acyl-CoA chain so that this reaction may occur (1).
VLCAD accepts longer acyl-Coa chains than MCAD because it has a larger binding cavity. The increased size of the cavity in comparison to MCAD is attributed to a salt-bridge between Arg-313 and Glu-354. Glu-354 is located on an alpha-helix which would otherwise cross the binding cavity, limiting the size of the cavity. The salt bridge between Arg-213 and Glu-354 holds the alpha-helix away from the binding site. A comparable salt-bridge is not present in MCAD (1).
VLCAD not only accepts longer chains, but thermodynamically prefers them. MCAD provides additional stabilization to the acyl-CoA chain via hydrogen bonding between Ser-166 and the thio-ester group of the acyl-Coa chain (2). In VLCAD, the beta-sheet on which the analogous serine residue ( Ser-181) is found extends past the catalytic site in VLCAD preventing similar stabilization. As a result, VLCAD depends on the stabilization provided by the interaction between the hydrocarbon chain and hydrophobic residues lining the cavity; therefore, the longer the hydrocarbon chain, the greater the stabilization of the substrate (1).
FAD binds primarily through hydrogen bonding or ionic interaction of residue side chains to its pyrophosphate group and adenosine group (6). Four interactions are particularly noteworthy. The first interaction involves a hydrogen bond between Ser-183 and an oxygen atom of the FAD pyrophosphate group. Ser-183 is located between two beta-strands on the same monomer as the FAD molecule it stabilizes. Mutations in the neighboring Gly-182 or Asp-184 substantially decrease the enzymes affinity for FAD by moving the location of the Ser-183 residue (6). The second interaction involves Phe-421. The way in which Phe-421 stabilizes FAD is not well documented, but it is reasonable to suggest that it stabilizes the riboflavin moiety of FAD via pi-electron interaction. A mutation in this residue prevents FAD from binding (4). Lastly, Thr-424 and Asp-426 form hydrogen bonds with the hydroxyl groups of adenosine (6).
Remarkably, interactions which stabilize FAD are often crucial for the stabilization and formation of quaternary structure. For example, a salt-bridge between Arg-326 of one monomer and the FAD pyrophosphate group of another monomer stabilizes FAD and the homodimer structure. A more complex association between three basic residues and the adenosine group of FAD has a similar function. Asp-426 forms a hydrogen bond with 2'-OH of the adenosine group of FAD. Asp-426 is located on the same monomer as the FAD which it stabilizes, but it also forms a salt-bridge with Lys-342 of the other monomer. Lys-342 is in turn stabilized by Glu-392 on the same monomer (6).
Unlike MCAD which is located in the mitochondrial matrix, VLCAD is monotopically bound to the inner-mitochondrial membrane (8). The additional 180 residue C-terminal domain in VLCAD accounts for this difference. A sequence of hydrophobic residues which accounts for VLCAD's ability to bind with the inner-mitochondrial membrane has not been found. Instead it is hypothesized that disordered residues 446-478 rearrange to form an amphipathic alpha-helix from residues 441 to 476 upon contact with the inner-mitochondrial membrane. The amphipathic alpha-helix inserts its hydrophobic face into the lipid bilayer of the luminal side of the mitochontdial membrane while leaving its hydrophillic face exposed to the mitochonrdial cytoplam in association with the polar head groups of the membrane. It is composed of six polar side chains on one face and an equal number of non-polar residues on the other (1). Mutation in a residue belonging to the amphipathic alpha-helix prevents VLCAD association with the inner-membrane (9).
Mutations in VLCAD may lead to cardiomyopathy, pericardial effusion, or intermittent hypoglycemia (10). VLCAD deficiency can be detected through genetic screening methods such as Tandem Mass Spectrometry. Current treatment focuses on alteration in diet. Pharmacological agents which interact with VLCAD directly are still under development (8).