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• W2043511247 abstract "The acyl-CoA dehydrogenases are a family of flavin adenine dinucleotide-containing enzymes that catalyze the first step in the β-oxidation of fatty acids and catabolism of some amino acids. They exhibit high sequence identity and yet are quite specific in their substrate binding. Short chain acyl-CoA dehydrogenase has maximal activity toward butyryl-CoA and negligible activity toward substrates longer than octanoyl-CoA. The crystal structure of rat short chain acyl-CoA dehydrogenase complexed with the inhibitor acetoacetyl-CoA has been determined at 2.25 Å resolution. Short chain acyl-CoA dehydrogenase is a homotetramer with a subunit mass of 43 kDa and crystallizes in the space group P321 with a = 143.61 Å and c = 77.46 Å. There are two monomers in the asymmetric unit. The overall structure of short chain acyl-CoA dehydrogenase is very similar to those of medium chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and bacterial short chain acyl-CoA dehydrogenase with a three-domain structure composed of N- and C-terminal α-helical domains separated by a β-sheet domain. Comparison to other acyl-CoA dehydrogenases has provided additional insight into the basis of substrate specificity and the nature of the oxidase activity in this enzyme family. Ten reported pathogenic human mutations and two polymorphisms have been mapped onto the structure of short chain acyl-CoA dehydrogenase. None of the mutations directly affect the binding cavity or intersubunit interactions. The acyl-CoA dehydrogenases are a family of flavin adenine dinucleotide-containing enzymes that catalyze the first step in the β-oxidation of fatty acids and catabolism of some amino acids. They exhibit high sequence identity and yet are quite specific in their substrate binding. Short chain acyl-CoA dehydrogenase has maximal activity toward butyryl-CoA and negligible activity toward substrates longer than octanoyl-CoA. The crystal structure of rat short chain acyl-CoA dehydrogenase complexed with the inhibitor acetoacetyl-CoA has been determined at 2.25 Å resolution. Short chain acyl-CoA dehydrogenase is a homotetramer with a subunit mass of 43 kDa and crystallizes in the space group P321 with a = 143.61 Å and c = 77.46 Å. There are two monomers in the asymmetric unit. The overall structure of short chain acyl-CoA dehydrogenase is very similar to those of medium chain acyl-CoA dehydrogenase, isovaleryl-CoA dehydrogenase, and bacterial short chain acyl-CoA dehydrogenase with a three-domain structure composed of N- and C-terminal α-helical domains separated by a β-sheet domain. Comparison to other acyl-CoA dehydrogenases has provided additional insight into the basis of substrate specificity and the nature of the oxidase activity in this enzyme family. Ten reported pathogenic human mutations and two polymorphisms have been mapped onto the structure of short chain acyl-CoA dehydrogenase. None of the mutations directly affect the binding cavity or intersubunit interactions. The mammalian acyl-CoA dehydrogenases (ACD) 1The abbreviations used are: ACDacyl-CoA dehydrogenaseFADflavin adenine dinucleotideSCADshort chain acyl-CoA dehydrogenasebSCADbacterial short chain acyl-CoA dehydrogenaseIVDisovaleryl-CoA dehydrogenaseMCADmedium chain acyl-CoA dehydrogenaseLCADlong chain acyl-CoA dehydrogenaseVLCADvery long chain acyl-CoA dehydrogenaseSBCADshort/branched chain acyl-CoA dehydrogenaseIBDisobutyryl-CoA dehydrogenaseGCADglutaryl-CoA dehydrogenaseCoAcoenzyme A are a family of homologous flavoproteins that are involved in mitochondrial fatty acid and amino acid metabolism. Current members include SCAD, MCAD, LCAD, VLCAD, IVD, SBCAD, IBD, and GCAD. A bacterial SCAD (bSCAD) has also been described in the Gram-positive anaerobe Megasphaera elsdenii. S-, M-, L-, and VLCAD are all involved in the oxidation of straight chain fatty acids, whereas IVD, SBCAD, IBD, and GCAD are involved in the catabolism of leucine, isoleucine, valine, and lysine, respectively (1.Ikeda Y. Dabrowski C. Tanaka K. J. Biol. Chem. 1983; 258: 1066-1076Abstract Full Text PDF PubMed Google Scholar, 2.Goodman S.I. Kratz L.E. Frerman F.E. Prog. Clin. Biol. Res. 1992; 375: 169-173PubMed Google Scholar, 3.Finocchiaro G. Ito M. Tanaka K. J. Biol. Chem. 1987; 262: 7982-7989Abstract Full Text PDF PubMed Google Scholar, 4.Matsubara Y. Indo Y. Naito E. Ozasa H. Glassberg R. Vockley J. Ikeda Y. Kraus J. Tanaka K. J. Biol. Chem. 1989; 264: 16321-16331Abstract Full Text PDF PubMed Google Scholar, 5.Rozen R. Vockley J. Zhou L. Milos R. Willard J. Fu K. Vicanek C. Low-Nang L. Torban E. Fournier B. Genomics. 1994; 24: 280-287Crossref PubMed Scopus (57) Google Scholar, 6.Beinert H. Boyer P.D. Lardy H. Myrback K. Enzymes. 2nd Ed. 7. Academic Press, New York1963: 447-466Google Scholar). All of these enzymes are soluble homotetramers with a subunit mass of ∼43 kDa and are components of the mitochondrial matrix, with the exception of VLCAD, which is a homodimer of subunit mass 73 kDa and is bound to the matrix face of the inner mitochondrial membrane (7.Ikeda Y. Ikeda K.O. Tanaka K. J. Biol. Chem. 1985; 260: 1311-1325Abstract Full Text PDF PubMed Google Scholar, 8.Hashimoto T. Coates P. Tanaka K. Peroxisomal and Mitochondrial Enzymes. New Developments in Fatty Acid Oxidation. Wiley-Liss, Inc., New York1992: 19-32Google Scholar). The ACDs catalyze the α,β-dehydrogenation of acyl-CoA thioesters, in which an α-hydrogen is abstracted as a proton from the acyl-CoA thioester substrate with a β-hydrogen transferred as a hydride ion to the N(5) position of the enzyme-bound FAD (9.Ghisla S. Engst S. Vock P. Kieweg V. Bross P. Nandy A. Rasched I. Strauss A. Yagi K. Flavins and Flavoproteins. Walter de Gruyter & Co, New York1993: 283-292Google Scholar, 10.Engel P. Muller F. Chemistry and Biochemistry of Flavoenzymes. CRC Press, Boca Raton1992: 597-655Google Scholar, 11.Pohl B. Raichle T. Ghisla S. Eur. J. Biochem. 1986; 160: 109-115Crossref PubMed Scopus (78) Google Scholar). The reduced ACD is then reoxidized by electron transfer flavoprotein in a series of two one-electron transfers (12.Crane F. Beinert H. J. Biol. Chem. 1956; 218: 717-731Abstract Full Text PDF PubMed Google Scholar). acyl-CoA dehydrogenase flavin adenine dinucleotide short chain acyl-CoA dehydrogenase bacterial short chain acyl-CoA dehydrogenase isovaleryl-CoA dehydrogenase medium chain acyl-CoA dehydrogenase long chain acyl-CoA dehydrogenase very long chain acyl-CoA dehydrogenase short/branched chain acyl-CoA dehydrogenase isobutyryl-CoA dehydrogenase glutaryl-CoA dehydrogenase coenzyme A The three-dimensional structures of MCAD, bSCAD, and IVD have already been reported (13.Kim J.-J.P. Wu J. Proc. Natl. Acad. Sci. U. S. A. 1988; 84: 6677-6681Crossref Scopus (82) Google Scholar, 14.Kim J.-J.P. Wang M. Paschke R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7523-7527Crossref PubMed Scopus (267) Google Scholar, 15.Djordjevic S. Pace C.P. Stankovich M.T. Kim J.-J.P. Biochemistry. 1995; 34: 2163-2171Crossref PubMed Scopus (96) Google Scholar, 16.Tiffany K.A. Roberts D.L. Wang M. Paschke R. Mohsen A.W. Vockley J. Kim J.-J.P. Biochemistry. 1997; 36: 8455-8464Crossref PubMed Scopus (94) Google Scholar). These data confirmed the homotetrameric nature of the enzyme and the identity of the catalytic glutamate that abstracts an α-hydrogen as a proton. Amino acid sequence alignments identified a common glutamate in SCAD and bSCAD at the same position. It was expected that the analogous Glu-368 residue in SCAD would be in position to act as the catalytic residue, which was confirmed by site-specific mutagenesis (17.Battaile K. Mohsen A.-W. Vockley J. Biochemistry. 1996; 35: 15356-15363Crossref PubMed Scopus (22) Google Scholar). Of particular interest in the ACD family is the nature of substrate specificity. The protein sequence similarity across all ACDs of ∼33% (4.Matsubara Y. Indo Y. Naito E. Ozasa H. Glassberg R. Vockley J. Ikeda Y. Kraus J. Tanaka K. J. Biol. Chem. 1989; 264: 16321-16331Abstract Full Text PDF PubMed Google Scholar) suggests a common substrate binding mode and reaction mechanism. Although each ACD has an optimal substrate, the pattern of specificity for an individual ACD can overlap with other ACDs (7.Ikeda Y. Ikeda K.O. Tanaka K. J. Biol. Chem. 1985; 260: 1311-1325Abstract Full Text PDF PubMed Google Scholar, 18.Ikeda Y. Tanaka K. J. Biol. Chem. 1983; 258: 9477-9487Abstract Full Text PDF PubMed Google Scholar, 19.Ikeda Y. Tanaka K. J. Biol. Chem. 1983; 258: 1077-1085Abstract Full Text PDF PubMed Google Scholar, 20.Lenich A.C. Goodman S.I. J. Biol. Chem. 1986; 261: 4090-4096Abstract Full Text PDF PubMed Google Scholar, 21.Williamson G. Engel P.C. Biochem. J. 1984; 218: 521-529Crossref PubMed Scopus (36) Google Scholar). Through mutagenesis (17.Battaile K. Mohsen A.-W. Vockley J. Biochemistry. 1996; 35: 15356-15363Crossref PubMed Scopus (22) Google Scholar, 22.Nandy A. Kieweg V. Krautle F.-G. Vock P. Kuchler B. Bross P. Kim J.-J.P. Rasched I. Ghisla S. Biochemistry. 1996; 35: 12402-12411Crossref PubMed Scopus (48) Google Scholar) and structural analysis (23.Lee H.-J.K. Wang M. Paschke R. Nandy A. Ghisla S. Kim J.-J.P. Biochemistry. 1996; 35: 12412-12420Crossref PubMed Scopus (70) Google Scholar), specific residues in the binding cavities of SCAD and MCAD have been studied to determine their roles in substrate specificity. Altering the position of the catalytic residue in SCAD and MCAD (Glu-368 and Glu-376, respectively) through mutagenesis to the corresponding position of the catalytic residue in LCAD/IVD (Glu-261 and Glu-254, respectively) slightly changed the activity profile of SCAD. The values of Vmax/Km for butyryl-CoA and octanoyl-CoA are 280 and 3.8 min−1μmol−1, respectively in the wild-type enzyme, whereas the corresponding values are 9 and 1.1 min−1μmol−1 in the E368G/G247E mutant. On the other hand, the analogous mutant in MCAD dramatically changed the specificity of MCAD from an optimal substrate of octanoyl-CoA to dodecanoyl-CoA. Although mutagenesis thus far has focused on the catalytic residue with the most success, clearly the amino acid residues in the binding cavity are of equal, if not greater, importance, as the minimal effect seen in the SCAD mutant study demonstrates. The deficiencies of all the ACDs combined represent an important group of metabolic disorders; however, only a few patients have been described with SCAD deficiency (24.Turnbull D.M. Bartlett K. Stevens D.L. Alberti K.G. Gibson G.J. Johnson M.A. McCulloch A.J. Sherratt H.S. N. Engl. J. Med. 1984; 311: 1232-1236Crossref PubMed Scopus (118) Google Scholar, 25.Amendt B. Green C. Sweetman L. Cloherty H. Shih V. Moon A. Teel L. Rhead W. J. Clin. Invest. 1987; 79: 1303-1309Crossref PubMed Scopus (146) Google Scholar, 26.Coates P.M. Hale D.E. Finocchiaro G. Tanaka K. Winter S. J. Clin. Invest. 1988; 81: 171-175Crossref PubMed Scopus (85) Google Scholar). SCAD deficiency can present in infants with an acute acidosis and myopathy or in adults with a chronic myopathy. Presentation typically involves muscle weakness, lethargy, free carnitine depletion with elevated plasma butyryl-carnitine, and ethylmalonic aciduria. Metabolic stress can result in the excretion of methylsuccinate. Several patients with SCAD deficiency have been reported as well as mutations thereof (27.Gregersen N. Winter V.S. Corydon M.J. Corydon T.J. Rinaldo P. Ribes A. Martinez G. Bennett M.J. Vianey-Saban C. Bhala A. Hale D.E. Lehnert W. Kmoch S. Roig M. Riudor E. Eiberg H. Andresen B.S. Bross P. Bolund L.A. Kolvraa S. Hum. Mol. Genet. 1998; 7: 619-627Crossref PubMed Scopus (119) Google Scholar, 28.Naito E. Indo Y. Tanaka K. J. Clin. Invest. 1990; 85: 1575-1582Crossref PubMed Scopus (68) Google Scholar, 29.Corydon M. Vockley J. Rinaldo P. Rhead W. Kjeldsen M. Winter V. Riggs C. Babovic-Vuksanovic D. Smeitink J. De Jong J. Levy H. Sewell A. Roe C. Matern D. Dasouki M. Gregersen N. Pediatr. Res. 2001; 49: 18-23Crossref PubMed Scopus (105) Google Scholar, 30.Kristensen M.J. Kmoch S. Bross P. Andresen B.S. Gregersen N. Hum. Mol. Genet. 1994; 3: 1711Crossref PubMed Scopus (20) Google Scholar). As the amino acid sequence identity between rat and human SCAD is 92%, we have mapped 10 reported clinical mutations and two polymorphisms onto the SCAD structure reported herein in an effort to gain insight into the mechanism of pathogenesis of SCAD deficiency. Here we report the three-dimensional structure of rat SCAD at 2.25Å resolution. Expression and purification of recombinant SCAD has been described elsewhere (17.Battaile K. Mohsen A.-W. Vockley J. Biochemistry. 1996; 35: 15356-15363Crossref PubMed Scopus (22) Google Scholar). Briefly, the mature coding region from the cDNA of SCAD was cloned into the isopropyl-1-thio-β-d-galactopyranoside-inducible expression vector pKK223-3 with the addition of an N-terminal methionine. After induction, Escherichia coli cultures were harvested by centrifugation and lysed by sonication. Recombinant SCAD was purified by successive chromatography steps including DE52 (Whatman), hydroxyapatite, and S-Sepharose (Amersham Biosciences). The enzyme was crystallized using the sitting drop vapor diffusion method (31.McPherson A. Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 182-187Google Scholar). Trigonal crystals with an orange tint grew from drops of 8.8 mg/ml SCAD in the presence of 1.2 molar equivalents of acetoacetyl-CoA/monomer of enzyme in 85 mm Tris acetate, pH 7.0, and 270 mm ammonium sulfate equilibrated against a solution containing 85 mm Tris acetate, pH 7.0, and 3.78m ammonium sulfate. The crystals belong to a trigonal space group with unit cell dimensions of a = 143.61 Å and c = 77.46 Å. Diffraction data were collected using a Rigaku RU-200 rotating anode x-ray generator operating at 50 kV and 100 mA with a 0.3-mm collimator coupled to an RAXIS-II detector system. Data reduction was carried out with the Denzo/Scalepack software package (32.Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38609) Google Scholar). Data collection and structure refinement statistics are given in Table I. The structure of SCAD was solved by the molecular replacement method using the software package X-PLOR (33.Brunger A. X-PLOR, Version 3.1. A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar) and the structure of MCAD (Protein Data Bank accession code 3MDD) as the probe. The initial probe structure was generated by manually replacing amino acid residues in MCAD to match the SCAD sequence. The highest peak from the cross-rotation function (θ1, θ2, θ3 = 274.78, 4.5, and 234.78°, respectively; 15–4 Å data with I/ς > 8) was used in the subsequent translation search. Two trigonal space groups, P321 and P312, were consistent with the systematic absence of extinction reflections. X-PLOR rigid body refinement based on the highest peak on the P321 translation search using reflections from 15 to 4 Å lowered the R-value from 43.7 to 36.1%. Several cycles of refinement with 6993 reflections with intensities of >4 ς (15–4 Å) yielded an R-value of 24.2%. At this point, all data with I/ς > 0 were used for the subsequent refinement using X-PLOR and later CNS (34.Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16979) Google Scholar), and manual adjustments were performed with the molecular graphics package TURBO (35.Roussel A. Cambillau C. TURBO-FRODO, Version 4.2. Bio-Graphics, Mountain View, CA1993Google Scholar). Water molecules were added in areas of electron density in difference Fourier maps that were <3.3 Å from a hydrogen bond partner. The final model containing 6070 non-hydrogen, non-solvent atoms and 260 water molecules yielded a final Rcryst of 16.0% (Rfree = 20.6%) (Protein Data Bank accession code 1JQI).Table IData collection and refinement statisticsData setSpace groupP321Unit cell (Å) a = b143.6c77.46Asymmetric unit content2 monomersRsym6.6%Total reflections113,237Number of independent reflections (I/ς > 0)36,527Resolution2.25 ÅCompleteness (%)83.1Final R-factor/R-free % (I/ς > 0)16.0/20.6Root-mean-square deviation from ideality Bond length (Å)0.006 Bond angle (°)1.2Protein atomsaNumber of non-hydrogen atoms.5856Ligand atomsaNumber of non-hydrogen atoms.108Cofactor atomsaNumber of non-hydrogen atoms.106Water molecules260a Number of non-hydrogen atoms. Open table in a new tab As expected from their sequence similarity, the overall structure of SCAD is similar to those of MCAD, IVD, and bSCAD. Like the structures of the other ACDs, the monomeric structure of SCAD is composed of three domains: an N-terminal α-helical domain containing six helices (A–F), a medial β-sheet domain containing seven β-strands (1.Ikeda Y. Dabrowski C. Tanaka K. J. Biol. Chem. 1983; 258: 1066-1076Abstract Full Text PDF PubMed Google Scholar, 2.Goodman S.I. Kratz L.E. Frerman F.E. Prog. Clin. Biol. Res. 1992; 375: 169-173PubMed Google Scholar, 3.Finocchiaro G. Ito M. Tanaka K. J. Biol. Chem. 1987; 262: 7982-7989Abstract Full Text PDF PubMed Google Scholar, 4.Matsubara Y. Indo Y. Naito E. Ozasa H. Glassberg R. Vockley J. Ikeda Y. Kraus J. Tanaka K. J. Biol. Chem. 1989; 264: 16321-16331Abstract Full Text PDF PubMed Google Scholar, 5.Rozen R. Vockley J. Zhou L. Milos R. Willard J. Fu K. Vicanek C. Low-Nang L. Torban E. Fournier B. Genomics. 1994; 24: 280-287Crossref PubMed Scopus (57) Google Scholar, 6.Beinert H. Boyer P.D. Lardy H. Myrback K. Enzymes. 2nd Ed. 7. Academic Press, New York1963: 447-466Google Scholar, 7.Ikeda Y. Ikeda K.O. Tanaka K. J. Biol. Chem. 1985; 260: 1311-1325Abstract Full Text PDF PubMed Google Scholar), and a C-terminal α-helical domain containing five helices (G–J in Fig. 1A). SCAD, like the other ACDs, forms a tetramer of identical monomers with 222 symmetry. The crystallographic asymmetric unit contains a dimer that forms the tetramer with a crystallographic 2-fold axis. The root-mean-square deviation for the α-carbon atoms between the two monomers in the asymmetric unit is 0.27Å. All but five residues were identified in the electron density map, the three N-terminal residues (Leu-1, His-2, Thr-3), the initiating methionine required for expressing the mature form of SCAD in a bacterial system, and Ser-388 at the C terminus. Fig. 1B shows an overlay of the Cα traces of one monomer each of SCAD, MCAD, IVD, and bSCAD. The average root-mean-square deviation between the Cα of SCAD and the other ACD structures ranges from 1.7 Å with MCAD and 1.5 Å with bSCAD to 1.2 Å with IVD. The structural alignment in Fig. 2 shows that all homologous strands and helices in the four structures have the same number of residues with the exception of the E-helix in which SCAD, IVD, and bSCAD have 21 residues compared with 20 in MCAD. The E-helix lines the “bottom” of the substrate binding cavity. This insertion (Ser-97 in SCAD) in the E-helix has the local effect of shifting its N-terminal two turns of the helix slightly closer to the binding cavity in SCAD/IVD/bSCAD and making the binding cavity more shallow. The length of loops is a primary difference in the structures, and yet most of these length differences do not appear to result in any significant difference in enzyme function. In the loops between β-strands 5 and 6, Pro-194 adopts a cis conformation.Figure 2Structure-based sequence alignment of the ACDs. The mature protein sequences of the four ACDs that had been subject to crystallographic studies were aligned manually by their secondary structural elements. The positions of the α-helices are indicated by the gray bars and the β-strands by the arrows above the sequence. The asterisk(*) indicates the position of the catalytic glutamate in bSCAD, pig MCAD, and rat SCAD. The caret (^) indicates the position of the catalytic glutamate in human IVD. Note that the catalytic residue of IVD is located >100 residues away from the other ACDs. The plus signs (+) indicate the positions of the reported mutations and polymorphisms in SCAD. Residues lining the binding cavity for the fatty-acyl portion of the substrate (from Table II) are shaded. GenPept sequence record numbers are: bSCAD, AAA03594; rat SCAD, AAA40669; human IVD, AAA52711; pig MCAD, AAA83759.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 3′-AMP moiety of the CoA is exposed to solvent, and the fatty acyl moiety is buried deeply inside the molecule near the isoalloxazine ring of the FAD. The binding cavities of SCAD, IVD, and bSCAD are more shallow than that of MCAD, which at the bottom expands away from the isoalloxazine ring of the FAD and ends near Glu-99. In the structure of MCAD without substrate, there are series of well defined water molecules in the substrate binding cavity. When the substrate binds in MCAD, water molecules are displaced, and the side chains of amino acids Gln-95, Glu-99, Leu-103, Val-259, Tyr-375, and Glu-376 move aside to accommodate the substrate (14.Kim J.-J.P. Wang M. Paschke R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7523-7527Crossref PubMed Scopus (267) Google Scholar). The movement of these amino acid side chains enlarges the cavity to accommodate substrate. Although structures of SCAD, bSCAD, and IVD without bound ligand have not been determined, it is reasonable to assume that water occupies the binding cavity in all ACDs in the absence of substrate, which is then displaced upon substrate binding. As the CoA portion of the substrate is common for all of the ACDs, it would be expected that the regions involved in binding the CoA moiety would be very similar. The backbones for all four dehydrogenases overlay very well at the top of the binding cavity with the exception of the loop between strands 4 and 5. This difference can be seen in the structural alignment in Fig. 2. Even though this region is different in the four structures, it does not appear to play a significant role in the binding of the CoA moiety to the enzyme. As in other ACD structures, adenosine binding is mediated mostly through interactions with a single monomer, particularly residues Asn-183, Lys-184, Phe-237, Lys-238, Met-241, Asp-245, and Thr-318 in SCAD (Fig. 3). The sole contribution to substrate binding from the adjacent monomer is through Phe-276, which forms a Van der Waals' interaction (3.81 Å) with the start of the pantothenic acid moiety. Asp-245 is conserved in all enzymes and forms a hydrogen bond with the C(6) amino group of the adenine. The shape of the cavity opening and the pantothene-binding portion of the cavity is uniformly shaped in all of the structures. A minor difference is in the opening of MCAD, which is slightly wider and extended in the direction of the adenosine moiety. Table II contains a list of structurally homologous residues in the ACDs that line the binding cavity for the fatty-acyl portion of the CoA thioester substrate. In addition, the isoalloxazine ring of the FAD forms one wall of the binding cavity in all ACDs. Eight residues are responsible for shaping the binding cavity of SCAD;Val-90, Val-94, Leu-98, Phe-128, Thr-163, Ile-251, Tyr-367, and the catalytic residue Glu-368 (Table II, Fig. 4A). The side chain of Ile-251 extends partially into the binding cavity and appears to be the critical residue for limiting the depth of the binding cavity. This is in part because of the altered trajectory of the G helix in SCAD compared with MCAD, a difference that was also observed in the bSCAD structure (15.Djordjevic S. Pace C.P. Stankovich M.T. Kim J.-J.P. Biochemistry. 1995; 34: 2163-2171Crossref PubMed Scopus (96) Google Scholar). In addition, the corresponding residue in MCAD is valine at this position, which adopts a conformation such that it does not interfere with the binding cavity. The small but significant activity toward octanoyl-CoA (Vmax/Km for octanoyl-CoA is 70-fold smaller than that of butyryl-CoA) of SCAD (17.Battaile K. Mohsen A.-W. Vockley J. Biochemistry. 1996; 35: 15356-15363Crossref PubMed Scopus (22) Google Scholar), suggesting that there is a certain degree of flexibility among the residues lining the binding cavity to allow longer substrates to bind. In bSCAD as in SCAD, eight residues shape the binding cavity, all of which are in the same position as SCAD and five of which are identical to those in SCAD: Ile-88, Ala-92, Leu-96, Phe-126, Thr-162, Val-250, Tyr-366, and the catalytic residue Glu-367 (Table II, Fig. 4B). In bSCAD, Ile-88 impinges on the binding cavity from the opposite side as Ile-251 does in SCAD. The homologous residue of Ile-251 in bSCAD is Val-250, which does not restrict the binding cavity. This is due to the direct approach of the Ile-88 side chain into the binding cavity (15.Djordjevic S. Pace C.P. Stankovich M.T. Kim J.-J.P. Biochemistry. 1995; 34: 2163-2171Crossref PubMed Scopus (96) Google Scholar) and the shorter side chain of the valine. As was the case with bSCAD, IVD also shares many similar residues lining the binding cavity with SCAD. The primary difference here is that the binding cavity in IVD is expanded laterally to accommodate branched chain substrates because of a glycine at position 374 (16.Tiffany K.A. Roberts D.L. Wang M. Paschke R. Mohsen A.W. Vockley J. Kim J.-J.P. Biochemistry. 1997; 36: 8455-8464Crossref PubMed Scopus (94) Google Scholar), which is a leucine in IBD and a tyrosine in other ACDs. Residues shaping the IVD binding cavity are Leu-95, Ala-99, Leu-103, Leu-133, Thr-168, Leu-258, Leu-370, Tyr-371, Gly-374, Ala-375, and the catalytic residue Glu-254 (Table II, Fig. 4C). Although bSCAD shared five identical residues with SCAD, IVD shares two. IVD has two residues in position to block the bottom of the binding cavity to longer chain substrates, Leu-258 (Ile-251 in SCAD) and Leu-95 (Val-90 in bSCAD) (16.Tiffany K.A. Roberts D.L. Wang M. Paschke R. Mohsen A.W. Vockley J. Kim J.-J.P. Biochemistry. 1997; 36: 8455-8464Crossref PubMed Scopus (94) Google Scholar). In MCAD, because of a larger binding cavity, more residues are involved in forming the binding cavity. These residues are Gln-95, Thr-96, Glu-99, Ala-100, Thr-102, Leu-103, Tyr-133, Thr-168, Thr-255, Pro-258, Val-259, Tyr-372, Tyr-375, and the catalytic residue Glu-376 (Table II, Fig. 4D). The increased size of the binding cavity of MCAD is due in part to the shorter amino acid side chains in MCAD of residues homologous to the blocking residues in SCAD/bSCAD/IVD and the difference in trajectory of the G- and E-helices that line the binding cavity. These two factors together allow for the expansion of the binding cavity away from the FAD, which allows for the binding of longer substrates.Table IIResidues lining the binding cavity in the four reported ACD structuresrSCADbSCADhIVDpMCADGln-95Val-90Ile-88Leu-95Thr-96Glu-99Val-94Ala-92Ala-99Ala-100Thr-102Leu-98Leu-96Leu-103Leu-103Phe-128Phe-126Leu-133Tyr-133Thr-163Thr-162Thr-168Thr-168Glu-254Thr-255Pro-258Ile-251Val-250Leu-258Val-259Leu-370Tyr-371Tyr-372Tyr-367Tyr-366Gly-374Tyr-375Glu-368Glu-367Ala-375Glu-376Residues in rows are structural homologs. The catalytic residues are in italics. Open table in a new tab Residues in rows are structural homologs. The catalytic residues are in italics. The binding of long acyl-CoAs to SCAD, bSCAD, and IVD is significantly affected because the side chains of Ile-251 (SCAD), Leu-258 (IVD) or Ile-88 (bSCAD) are blocking the bottom of the binding cavity. The insertion of an extra residue in helix E results in the bending of the helix toward the active site in SCAD/bSCAD/IVD. In MCAD, on the other hand, a proline substitution in the G-helix makes the helix bend away from the active site. For SCAD and IVD the blocking residue (Ile-251/Leu-258) is on one side of the binding cavity, whereas for bSCAD it (Ile-88) is on the other side. The second structural difference appears to be the position of the peptide backbone in the region of the binding cavity. All of the enzymes have subtle differences in the position of the main chain atoms. In the vicinity of the binding cavity, these differences result in the movement of amino acid side chains further into the binding cavity, as is the case with SCAD/bSCAD/IVD, or further away from the binding cavity, as is the case in MCAD and, presumably, LCAD/VLCAD. The combination of the differences in the residues, combined with the trajectory of the helices they are part of, together help to modulate the size of the bottom of the binding cavity, thus controlling the binding of longer substrates. The molecular basis of oxygen reactivity of ACDs has been a subject of some debate (36.Johnson J.K. Kumar N.R. Srivastava D.K. Biochemistry. 1994; 33: 4738-4744Crossref PubMed Scopus (21) Google Scholar, 37.DuPlessis E.R. Pellett J. Stankovich M.T. Thorpe C. Biochemistry. 1998; 37: 10469-10477Crossref PubMed Scopus (30) Google Scholar, 38.Wang R. Thorpe C. Biochemistry. 1991; 30: 7895-7901Crossref PubMed Scopus (68) Google Scholar). When reduced flavins of mammalian ACDs are bound to certain acyl-CoA ligands, reoxidation by molecular oxygen of the flavins is dramatically slowed. Wang and Thorpe (38.Wang R. Thorpe C. Biochemistry. 1991; 30: 7895-7901Crossref PubMed Scopus (68) Google Scholar) showed that the half-life of the photo-reduced FAD of pig MCAD was increased up to 3,600-fold when complexed with certain substrate analogs. Rat SCAD also shows a similar result when bound to substrate/product analogs. 2M. S" @default.
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• W2043511247 date "2002-04-01" @default.
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• W2043511247 title "Crystal Structure of Rat Short Chain Acyl-CoA Dehydrogenase Complexed with Acetoacetyl-CoA" @default.
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