Structural Basis for Substrate Fatty Acyl Chain Specificity

Abstract

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a member of the family of acyl-CoA dehydrogenases (ACADs). Unlike the other ACADs, which are soluble homotetramers, VLCAD is a homodimer associated with the mitochondrial membrane. VLCAD also possesses an additional 180 residues in the C terminus that are not present in the other ACADs. We have determined the crystal structure of VLCAD complexed with myristoyl-CoA, obtained by co-crystallization, to 1.91-Å resolution. The overall fold of the N-terminal ϳ400 residues of VLCAD is similar to that of the soluble ACADs including medium-chain acyl-CoA dehydrogenase (MCAD). The novel C-terminal domain forms an ␣-helical bundle that is positioned perpendicular to the two N-terminal helical domains. The fatty acyl moiety of the bound substrate/product is deeply imbedded inside the protein; however, the adenosine pyrophosphate portion of the C14-CoA ligand is disordered because of partial hydrolysis of the thioester bond and high mobility of the CoA moiety. The location of Glu-422 with respect to the C2-C3 of the bound ligand and FAD confirms Glu-422 to be the catalytic base. In MCAD, Gln-95 and Glu-99 form the base of the substrate binding cavity. In VLCAD, these residues are glycines (Gly-175 and Gly-178), allowing the binding channel to extend for an additional 12 Å and permitting substrate acyl chain lengths as long as 24 carbons to bind. VLCAD deficiency is among the more common defects of mitochondrial ␤-oxidation and, if left undiagnosed, can be fatal. This structure allows us to gain insight into how a variant VLCAD genotype results in a clinical phenotype. Very-long-chain acyl-CoA dehydrogenase (VLCAD)3 is one of five acyl-CoA dehydrogenases (ACADs) that catalyze the initial, rate-limiting step of mitochondrial fatty acid ␤-oxidation, with distinct but overlapping fatty acyl chain-length specificities (1, 2). In addition to VLCAD, which has optimal chain length specificity for fatty acyl-CoAs having 16 carbons in length, there are long-, medium-, and short-chain acyl-CoA dehydrogenases (LCAD, MCAD, and SCAD), which are most active with 14, 8, and 4 carbon substrates, respectively (3, 4). In addition, acyl-CoA dehydrogenase 9 (ACAD-9) is most active with unsaturated long-chain acyl-CoAs (5). The ACAD family also includes four members involved in amino acid metabolic pathways: isobutyryl-CoA dehydrogenase (IBD) in valine metabolism, isovaleryl-CoA dehydrogenase (IVD) in leucine metabolism, glutaryl-CoA dehydrogenase (GCAD) in lysine and tryptophan metabolism, and short-branched chain acylCoA dehydrogenase (SBCAD) in isoleucine metabolism. Fatty acyl-CoAs are oxidized to the corresponding trans-2,3-enoylCoA products with a concurrent reduction of the enzymebound FAD cofactor (6). Electron transfer flavoprotein (ETF) reoxidizes the reduced flavin and transfers reducing equivalents to the main mitochondrial respiratory chain through the enzyme ETF-ubiquinone oxidoreductase (7). Unlike other ACADs, which are soluble homotetramers with 45-kDa subunits, mature VLCAD and A...

VLCAD enzyme activity determinations in newborns identified by screening: a valuable tool for risk assessment

“…We delineated 21 different mutations in 24 subjects and classified 16 missense mutations and five null mutations (Table 1). We used the 3D crystal structure of VLCAD first described by Souri (Souri et al 1998) and confirmed by McAndrew (McAndrew et al 2008) to locate the mutations and correlate their predicted effect on enzyme stability as functionally measured by palmitoyl-CoA oxidation in lymphocytes.…”

“…The crystal structure of the human VLCAD enzyme was published by McAndrew (McAndrew et al 2008) and can be reviewed in Pubmed (MMDB ID 62408, PDB ID 3B96). Tube models of the human VLCAD monomer have been generated using the Cn3D 4.1 software provided by the National Center of Biotechnology Information (NCBI).…”

Purification, crystallization and preliminary crystallographic analysis of very-long-chain acyl-CoA dehydrogenase fromCaenorhabditis elegans

“…All of these soluble tetrameric ACADs exhibit 'dimer-ofdimer' structures and all their subunits possess a similar fold consisting of an N-terminal -helical domain, a middle -sheet domain and a C-terminal -helical domain. The crystal structure of human VLCAD (hVLCAD) complexed with myristoyl-CoA was first determined at 1.91 Å resolution and the overall fold of its N-terminal $400 residues is similar to those of other soluble ACADs; its additional C-terminal 180 residues are proposed to be involved in binding to the membrane (McAndrew et al, 2008).…”

Evidence for the physical association of mitochondrial fatty acid oxidation and oxidative phosphorylation complexes

“…ETF and ETF dehydrogenase have been shown to form a stable complex, and long chain 3-hydroxyacyl-CoA dehydrogenase has been shown to interact directly with ETC complex I at the inner mitochondria membrane (14 -16). Finally, we have recently shown that very long chain acyl-CoA dehydrogenase (a member of the ACAD gene family) interacts directly with the mitochondrial membrane in a way that provides a possible direct physical connection between FAO and OXPHOS (17,18).…”