The three-dimensional strcture of mediumchain acyl-CoA dehydrogenase from pig mitochondria in the native form and that ofa complex ofthe enzyme and a strate (product) have been solved and refined by x-ray crystallo- Mammalian acyl-CoA dehydrogenases [acyl-CoA:(acceptor) 2,3-oxidoreductase; EC 1.3.99.3] catalyze the first step in each cycle of fatty acid a-oxidation in mitochondria (1).Acyl-CoA thioesters are oxidized to the corresponding trans-2,3-enoyl-CoA products with concomitant reduction of enzyme-bound FAD. Reoxidation of the dehydrogenase flavin and the transfer of reducing equivalents to the mitochondrial respiratory chain are catalyzed by the soluble electron transferring flavoprotein (ETF) and the particulate ETF-ubiquinone oxidoreductase, an iron-sulfur flavoprotein (2, 3).Three soluble stright-chain acyl-CoA dehydrogenases have been isolated and classified according to their distinct but overlapping substrate specificities for long-, medium-, and short-chain fatty acids (4). Recently, a very-long-chain acylCoA dehydrogenase has been identified in the inner membrane of rat mitochondria (5). In addition, three dehydrogenases involved in amino acid metabolism, isovaleryl-(6), 2-methyl branched chain (7), and glutaryl-(8) CoA dehydrogenases, have been isolated and characterized. With the exception of the membrane-associated very-long-chain acylCoA dehydrogenase, these enzymes appear very similar in their catalytic mechanism and their biochemical properties.They are homotetramers and each subunit contains --400 aaThe publication costs of this article were defrayed in part by page chare payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. residues and one equivalent of FAD. Medium-chain acylCoA dehydrogenase (MCAD) exhibits a broad chain-length specificity and has its highest activity with Cg-CoA. Re-cently, several genetic diseases have been found to be due to acyl-CoA dehydrogenase deficiencies. Deficiency of MCAD appears to be the most common among disorders offatty acid oxidation in humans (9). It is manifested by fasting intolerance, hypoglycemic coma, failure of ketogenesis, dicarboxylic acidemia, and sudden infant death (10). Although the enzyme structure described in this report is that of the porcine liver enzyme, the amino acid sequence is very similar to that of the human enzyme (11), from which it differs by <10% conservative substitutions (A. W. Strauss, personal communication). Therefore, it is expected that structural conclusions drawn from the pig enzyme will apply to the human enzyme. We have reported the crystal structure of pig-liver MCAD at 3.0-A resolution (12) and preliminary data on complexes of the enzyme and substrates (13). In this paper, we present the crystal structure ofMCAD in the native form and that of a complex of the enzyme and the octanoylCoA, both refined at 2.4-A resolution.

Acyl-CoA dehydrogenases and acyl-CoA oxidases are two closely related FAD-containing enzyme families that are present in mitochondria and peroxisomes, respectively. They catalyze the dehydrogenation of acyl-CoA thioesters to the corresponding trans-2-enoyl-CoA. This review examines the structure of medium chain acyl-CoA dehydrogenase, as a representative of the dehydrogenase family, with respect to the catalytic mechanism and its broad chain length specificity. Comparing the structures of four other acyl-CoA dehydrogenases provides further insights into the structural basis for the substrate specificity of each of these enzymes. In addition, the structure of peroxisomal acyl-CoA oxidase II from rat liver is compared to that of medium chain acyl-CoA dehydrogenase, and the structural basis for their different oxidative half reactions is discussed.

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.

Mammalian electron transfer f lavoproteins (ETF) are heterodimers containing a single equivalent of f lavin adenine dinucleotide (FAD). They function as electron shuttles between primary f lavoprotein dehydrogenases involved in mitochondrial fatty acid and amino acid catabolism and the membrane-bound electron transfer flavoprotein ubiquinone oxidoreductase. The structure of human ETF solved to 2.1-Å resolution reveals that the ETF molecule is comprised of three distinct domains: two domains are contributed by the ␣ subunit and the third domain is made up entirely by the ␤ subunit. The N-terminal portion of the ␣ subunit and the majority of the ␤ subunit have identical polypeptide folds, in the absence of any sequence homology. FAD lies in a cleft between the two subunits, with most of the FAD molecule residing in the C-terminal portion of the ␣ subunit. Alignment of all the known sequences for the ETF ␣ subunits together with the putative FixB gene product shows that the residues directly involved in FAD binding are conserved. A hydrogen bond is formed between the N5 of the FAD isoalloxazine ring and the hydroxyl side chain of ␣T266, suggesting why the pathogenic mutation, ␣T266M, affects ETF activity in patients with glutaric acidemia type II. Hydrogen bonds between the 4-hydroxyl of the ribityl chain of FAD and N1 of the isoalloxazine ring, and between ␣H286 and the C2-carbonyl oxygen of the isoalloxazine ring, may play a role in the stabilization of the anionic semiquinone. With the known structure of medium chain acyl-CoA dehydrogenase, we hypothesize a possible structure for docking the two proteins.

Mevalonate kinase catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the pathways of isoprenoids and sterols. Deficiency in mevalonate kinase activity has been linked to mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS). The crystal structure of rat mevalonate kinase in complex with MgATP has been determined at 2.4-Å resolution. Each monomer of this dimeric protein is composed of two domains with its active site located at the domain interface. The enzyme-bound ATP adopts an anti conformation, in contrast to the syn conformation reported for Methanococcus jannaschii homoserine kinase. The Mg 2؉ ion is coordinated to both ␤-and ␥-phosphates of ATP and side chains of Glu 193 and Ser 146 . Asp 204 is making a salt bridge with Lys 13 , which in turn interacts with the ␥-phosphate. A model of mevalonic acid can be placed near the ␥-phosphoryl group of ATP; thus, the C5 hydroxyl is located within 4 Å from Asp 204 , Lys 13 , and the ␥-phosphoryl of ATP. This arrangement of residues strongly suggests: 1) Asp 204 abstracts the proton from C5 hydroxyl of mevalonate; 2) the penta-coordinated ␥-phosphoryl group may be stabilized by Mg 2؉ , Lys 13 , and Glu 193 ; and 3) Lys 13 is likely to influence the pK a of the C5 hydroxyl of the substrate. V377I and I268T are the most common mutations found in patients with HIDS. Val 377 is located over 18 Å away from the active site and a conservative replacement with Ile is unlikely to yield an inactive or unstable protein. Ile-268 is located at the dimer interface, and its Thr substitution may disrupt dimer formation.Mevalonate kinase (MK, ATP:mevalonate 5-phosphotransferase, EC 2.7.1.36) 1 catalyzes the transfer of the ␥-phosphoryl group from ATP to the C5 hydroxyl oxygen of mevalonic acid to form mevalonate 5-phosphate, a key intermediate in the biosynthetic pathway for isoprenoids and sterols from acetate. Although the enzyme was discovered in the late 1950s (1, 2), it suffered over three decades of neglect, as research on the isoprenoid pathway was focused on HMG-CoA reductase, which catalyzes the previous step in the pathway, i.e. the formation of mevalonic acid from HMG-CoA. However, interest in MK has been revived recently, because it was recognized that this enzyme, together with HMG-CoA synthase and HMG-CoA reductase, is involved in coordinate regulation of this pathway and therefore may represent a secondary control point. The recent recognition of the involvement of the diverse non-sterol isoprenoid metabolites in various cellular functions (e.g. protein prenylation, protein glycosylation, and cell cycle regulation) has also increased interest in this enzyme. In addition, the significance of MK has been further highlighted by the implication of the enzyme in human inherited diseases, such as mevalonic aciduria and hyperimmunoglobulinemia D/periodic fever syndrome (HIDS, Mendelian Inheritance in Man 260920).The enzyme is found in eukaryotes, archaebacteria, and some eubacteria. Th...

Electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is a 4Fe4S flavoprotein located in the inner mitochondrial membrane. It catalyzes ubiquinone (UQ) reduction by ETF, linking oxidation of fatty acids and some amino acids to the mitochondrial respiratory chain. Deficiencies in ETF or ETF-QO result in multiple acyl-CoA dehydrogenase deficiency, a human metabolic disease. Crystal structures of ETF-QO with and without bound UQ were determined, and they are essentially identical. The molecule forms a single structural domain. Three functional regions bind FAD, the 4Fe4S cluster, and UQ and are closely packed and share structural elements, resulting in no discrete structural domains. The UQbinding pocket consists mainly of hydrophobic residues, and UQ binding differs from that of other UQ-binding proteins. ETF-QO is a monotopic integral membrane protein. The putative membranebinding surface contains an ␣-helix and a ␤-hairpin, forming a hydrophobic plateau. The UQOflavin distance (8.5 Å) is shorter than the UQOcluster distance (18.8 Å), and the very similar redox potentials of FAD and the cluster strongly suggest that the flavin, not the cluster, transfers electrons to UQ. Two possible electron transfer paths can be envisioned. First, electrons from the ETF flavin semiquinone may enter the ETF-QO flavin one by one, followed by rapid equilibration with the cluster. Alternatively, electrons may enter via the cluster, followed by equilibration between centers. In both cases, when ETF-QO is reduced to a two-electron reduced state (one electron at each redox center), the enzyme is primed to reduce UQ to ubiquinol via FAD.fatty acid oxidation ͉ iron-sulfur flavoprotein ͉ mitochondrial respiratory chain ͉ membrane protein ͉ acyl-CoA dehydrogenases E lectron transfer f lavoprotein-ubiquinone oxidoreductase (ETF-QO) is an intrinsic membrane protein located in the inner mitochondrial membrane. It contains single equivalents of FAD and a [4Fe4S] 2ϩ,1ϩ cluster (1). The protein is the single input site to the main respiratory chain for electrons from nine flavoprotein acyl-CoA dehydrogenases and two N-methyl dehydrogenases (2, 3). The electron acceptor for the dehydrogenases is the ETF, which is the reductant of ETF-QO. ETF-QO is oxidized by the diffusible ubiquinone (UQ) pool that also is accessed by NADH-UQ oxidoreductase (Complex I), succinate-UQ oxidoreductase (Complex II), the flavin-linked glycerol-3-phosphate dehydrogenase, and dihydroorotate dehydrogenase, another flavin-linked UQ oxidoreductase (4). The ubiquinol product of these oxidoreductases transfers electrons to the bc 1 complex (Complex III). Thus, ETF and ETF-QO link the oxidation of fatty acids and some amino acids to the mitochondrial respiratory system, and the overall electron flow can be summarized as follows: Acyl-CoA 3 Acyl-CoA dehydrogenases 3 ETF 3 ETF-QO 3 UQ 3 Complex III. Inherited deficiencies of ETF-QO or ETF cause a metabolic disease, multiple acyl-CoA dehydrogenase deficiency, also known as glutaric acidemia type II (5). This metab...

Botulinum neurotoxin causes rapid flaccid paralysis through inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside- protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that of HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using α(1-3,4)- fucosidase, endo-β-galactosidase and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain α2, 3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order: GT1b = GD1a ≫ GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for neurons.

Tetanus neurotoxin (TeNT) is an exotoxin produced byClostridium tetani that causes paralytic death to hundreds of thousands of humans annually. TeNT cleaves vesicle-associated membrane protein-2, which inhibits neurotransmitter release in the central nervous system to elicit spastic paralysis, but the molecular basis for TeNT entry into neurons remains unclear. TeNT is a ϳ150-kDa protein that has AB structure-function properties; the A domain is a zinc metalloprotease, and the B domain encodes a translocation domain and C-terminal receptor-binding domain (HCR/T). Earlier studies showed that HCR/T bound gangliosides via two carbohydrate-binding sites, termed the lactose-binding site (the "W" pocket) and the sialic acid-binding site (the "R" pocket). Here we report that TeNT high affinity binding to neurons is mediated solely by gangliosides. Glycan array and solid phase binding analyses identified gangliosides that bound exclusively to either the W pocket or the R pocket of TeNT; GM1a bound to the W pocket, and GD3 bound to the R pocket. Using these gangliosides and mutated forms of HCR/T that lacked one or both carbohydrate-binding pocket, gangliosides binding to both of the W and R pockets were shown to be necessary for high affinity binding to neuronal and non-neuronal cells. The crystal structure of a ternary complex of HCR/T with sugar components of two gangliosides bound to the W and R supported the binding of gangliosides to both carbohydrate pockets. These data show that gangliosides are functional dual receptors for TeNT.Tetanus is an acute, often fatal disease of humans that was first described by Hippocrates over 24 centuries ago (1). Tetanus is characterized by generalized increased rigidity and convulsive spasms of skeletal muscles. Tetanus is caused by exposure to tetanus neurotoxin (TeNT) 3 produced by the sporeforming bacterium Clostridium tetani. TeNT is delivered from the bloodstream to the peripheral nervous system, from where TeNT traffics to the central nervous system to cleave vesicleassociated membrane protein-2 (VAMP2), which inhibits neurotransmitter release and elicits spastic paralysis (2). Although prevented by vaccination, tetanus is responsible for hundreds of thousands of deaths per year in countries where vaccination is not common (3).TeNT is produced as a ϳ150-kDa protein that is cleaved to a di-chain protein, comprising an N-terminal light chain (ϳ50 kDa) and a C-terminal heavy chain domain (ϳ100 kDa) linked through a single disulfide bond (4). TeNT light chain is a zinc metalloprotease that cleaves the neuronal SNARE protein VAMP2 (2). The TeNT heavy chain contains two functional domains: a translocation domain and a C-terminal receptorbinding domain (HCR/T, ϳ50 kDa).The first step in TeNT action involves binding to a receptor(s) on the presynaptic membrane of ␣-motor neurons. Although the molecular basis for TeNT entry remains undetermined, an unambiguous role for gangliosides has been demonstrated (5-9). Current models implicate a dual receptor mechanism for the binding of t...