FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1985134465> ?p ?o ?g. }

• W1985134465 endingPage "28487" @default.
• W1985134465 startingPage "28480" @default.
• W1985134465 abstract "Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general. Carnitine acyltransferases catalyze the reversible exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids in the mitochondria for energy production, and are attractive targets for drug discovery against diabetes and obesity. To help define in molecular detail the catalytic mechanism of these enzymes, we report here the high resolution crystal structure of wild-type murine carnitine acetyltransferase (CrAT) in a ternary complex with its substrates acetyl-CoA and carnitine, and the structure of the S554A/M564G double mutant in a ternary complex with the substrates CoA and hexanoylcarnitine. Detailed analyses suggest that these structures may be good mimics for the Michaelis complexes for the forward and reverse reactions of the enzyme, representing the first time that such complexes of CrAT have been studied in molecular detail. The structural information provides significant new insights into the catalytic mechanism of CrAT and possibly carnitine acyltransferases in general. Carnitine acyltransferases catalyze the reversible exchange of acyl groups between carnitine and coenzyme A (CoA) 3The abbreviations used are: CoA, coenzyme A; CrAT, carnitine acetyltransferase; CrOT, carnitine octanoyltransferase; CPT, carnitine palmitoyltransferase; PEG, polyethylene glycol; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.3The abbreviations used are: CoA, coenzyme A; CrAT, carnitine acetyltransferase; CrOT, carnitine octanoyltransferase; CPT, carnitine palmitoyltransferase; PEG, polyethylene glycol; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol. (1Bieber L.L. Annu. Rev. Biochem. 1988; 57: 261-283Crossref PubMed Scopus (667) Google Scholar, 2Foster D.W. Ann. N. Y. Acad. Sci. 2004; 1033: 1-16Crossref PubMed Scopus (166) Google Scholar, 3Colucci W.J. Gandour R.D. Bioorg. Chem. 1988; 16: 307-334Crossref Scopus (75) Google Scholar, 4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 6Bonnefont J.P. Djouadi F. Prip-Buus C. Gobin S. Munnich A. Bastin J. Mol. Aspects Med. 2004; 25: 495-520Crossref PubMed Scopus (424) Google Scholar, 7Steiber A. Kerner J. Hoppel C.L. Mol. Aspects Med. 2004; 25: 455-473Crossref PubMed Scopus (307) Google Scholar, 8Ramsay R.R. Zammit V.A. Mol. Aspects Med. 2004; 25: 475-493Crossref PubMed Scopus (120) Google Scholar). These enzymes are classified based on their preference for the chain length of the acyl groups. Carnitine acetyltransferase (CrAT) and carnitine octanoyltransferase (CrOT) prefer short- and medium-chain acyl groups as substrates, respectively. Carnitine palmitoyltransferases (CPTs) prefer long-chan acyl groups as substrates. Several different isoforms of CPTs have been identified in mammals, including CPT-Ia (liver isoform), CPT-Ib (muscle isoform), CPT-Ic (brain isoform), and CPT-II. The CPT-Is are integrally associated with the outer membrane of the mitochondria, whereas CPT-II is localized in the mitochondrial matrix and may be loosely associated with the inner membrane of the mitochondria.The CPTs catalyze the rate-limiting step in the β-oxidation of fatty acids in the mitochondria (4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 6Bonnefont J.P. Djouadi F. Prip-Buus C. Gobin S. Munnich A. Bastin J. Mol. Aspects Med. 2004; 25: 495-520Crossref PubMed Scopus (424) Google Scholar, 7Steiber A. Kerner J. Hoppel C.L. Mol. Aspects Med. 2004; 25: 455-473Crossref PubMed Scopus (307) Google Scholar, 8Ramsay R.R. Zammit V.A. Mol. Aspects Med. 2004; 25: 475-493Crossref PubMed Scopus (120) Google Scholar, 9Kerner J. Hoppel C. Biochim. Biophys. Acta. 2000; 1486: 1-17Crossref PubMed Scopus (583) Google Scholar), which is the transport of fatty acids from the cytosol into the mitochondria. The CoA esters of fatty acids cannot cross the mitochondrial membranes. Instead, they must be converted to their carnitine esters, a reaction that is catalyzed by the CPT-Is, which can then be transported into the mitochondria. Once inside, the carnitine esters are converted back to the CoA esters through the action of CPT-II. Mutation and dysregulation of the CPTs are strongly linked to many serious, even fatal human diseases (4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 6Bonnefont J.P. Djouadi F. Prip-Buus C. Gobin S. Munnich A. Bastin J. Mol. Aspects Med. 2004; 25: 495-520Crossref PubMed Scopus (424) Google Scholar). CPT deficiencies are among the most common causes of inherited fatty acid oxidation disorders, and homozygous deletion of CPT-1a is lethal in mice (10Nyman L.R. Cox K.B. Hoppel C.L. Kerner J. Barnoski B.L. Hamm D.A. Tian L. Schoeb T.R. Wood P.A. Mol. Gen. Metab. 2005; 86: 179-187Crossref PubMed Scopus (52) Google Scholar). Inhibitors of CPT-Is may be efficacious for the treatment of type 2 diabetes (11Anderson R.C. Curr. Pharm. Des. 1998; 4: 1-16PubMed Google Scholar, 12Lenhard J.M. Gottschalk W.K. Adv. Drug Delivery Rev. 2002; 54: 1199-1212Crossref PubMed Scopus (41) Google Scholar), whereas agonists of these enzymes can stimulate fatty acid oxidation and may regulate body weight (13Thupari J.N. Landree L.E. Ronnett G.V. Kuhajda F.P. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9498-9502Crossref PubMed Scopus (212) Google Scholar, 14Ronnett G.V. Kim E.-K. Landree L.E. Tu Y. Physiol. Behav. 2005; 85: 25-35Crossref PubMed Scopus (101) Google Scholar). CPT-I has also been proposed as a target for the treatment of heart failure (15Mengi S.A. Dhalla N.S. Am. J. Cardiovasc. Drugs. 2004; 4: 201-209Crossref PubMed Scopus (16) Google Scholar).The CrAT and CrOT enzymes may be important for the oxidation and transport of fatty acids from the peroxisomes to the mitochondria (4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 7Steiber A. Kerner J. Hoppel C.L. Mol. Aspects Med. 2004; 25: 455-473Crossref PubMed Scopus (307) Google Scholar, 8Ramsay R.R. Zammit V.A. Mol. Aspects Med. 2004; 25: 475-493Crossref PubMed Scopus (120) Google Scholar). CrAT may also have an important role in maintaining the CoA/acetyl-CoA balance in the cells (4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar, 7Steiber A. Kerner J. Hoppel C.L. Mol. Aspects Med. 2004; 25: 455-473Crossref PubMed Scopus (307) Google Scholar, 8Ramsay R.R. Zammit V.A. Mol. Aspects Med. 2004; 25: 475-493Crossref PubMed Scopus (120) Google Scholar).The catalytic domains of carnitine acyltransferases contain about 600 amino acid residues and share significant sequence identity (35% and higher). The CPT-Is contain an N-terminal extension of about 140 residues that are important for attachment to the mitochondrial membrane and other functions. We and others have reported the crystal structures of CrAT (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 17Hsiao Y.-S. Jogl G. Tong L. J. Biol. Chem. 2004; 279: 31584-31589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 18Wu D. Govindasamy L. Lian W. Gu Y. Kukar T. Agbandje-McKenna M. McKenna R. J. Biol. Chem. 2003; 278: 13159-13165Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 19Ramsay R.R. Naismith J.H. Trends Biochem. Sci. 2003; 28: 343-346Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 20Govindasamy L. Kukar T. Lian W. Pedersen B. Gu Y. Agbandje-McKenna M. Jin S. McKenna R. Wu D. J. Struct. Biol. 2004; 146: 416-424Crossref PubMed Scopus (17) Google Scholar, 21Jogl G. Hsiao Y.-S. Tong L. Ann. N. Y. Acad. Sci. 2004; 1033: 17-29Crossref PubMed Scopus (99) Google Scholar), CrOT (22Jogl G. Hsiao Y.-S. Tong L. J. Biol. Chem. 2005; 280: 738-744Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), CPT-II (23Rufer A.C. Thoma R. Benz J. Stihle M. Gsell B. de Roo E. Banner D.W. Mueller F. Chomienne O. Hennig M. Structure. 2006; 14: 713-723Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 24Hsiao Y.-S. Jogl G. Esser V. Tong L. Biochem. Biophys. Res. Commun. 2006; 346: 974-980Crossref PubMed Scopus (34) Google Scholar), and the related enzyme choline acetyltransferase (25Cai Y. Cronin C.N. Engel A.G. Ohno K. Hersh L.B. Rodgers D.W. EMBO J. 2004; 23: 2047-2058Crossref PubMed Scopus (49) Google Scholar, 26Govindasamy L. Pedersen B. Lian W. Kukar T. Gu Y. Jin S. Agbandje-McKenna M. Wu D. McKenna R. J. Struct. Biol. 2004; 148: 226-235Crossref PubMed Scopus (25) Google Scholar). The structures can be divided into two domains, N and C domains (Fig. 1), that share the same backbone fold with that of chloramphenicol acetyltransferase and several other acetyltransferases (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 27Leslie A.G.W. Moody P.C. Shaw W.V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4133-4137Crossref PubMed Scopus (132) Google Scholar).The active site of the enzyme is located at the interface between the two domains, and the catalytic His residue is at the center of a tunnel that extends through the middle of the enzyme (Fig. 1) (21Jogl G. Hsiao Y.-S. Tong L. Ann. N. Y. Acad. Sci. 2004; 1033: 17-29Crossref PubMed Scopus (99) Google Scholar). Structures of the binary complexes with carnitine or CoA have defined the binding modes of these substrates and suggested a catalytic mechanism in which the positive charge on the carnitine molecule helps to stabilize the oxyanion of the transition state (substrate-assisted catalysis) (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Additional stabilization of the oxyanion is provided by hydrogen bonding to the side chain of a strictly conserved Ser residue (Ser554 in CrAT).To further probe the catalytic mechanism, it is desirable to obtain structural information on the ternary complexes of this enzyme with its substrates or products. Alternatively, structures of the enzyme in complex with bisubstrate analogs could be used to provide insight on the catalysis, as has been carried out successfully for N-acetyltransferases (28Scheibner K.A. De Angelis J. Burley S.K. Cole P.A. J. Biol. Chem. 2002; 277: 18118-18126Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 29Hickman A.B. Namboodiri M.A. Klein D.C. Dyda F. Cell. 1999; 97: 361-369Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). However, such compounds are not readily available for CrAT, and we have therefore developed protocols for trapping the ternary complexes of this enzyme. We report here the crystal structures of wild-type mouse CrAT in a ternary (Michaelis) complex with the substrates acetyl-CoA and carnitine and in a ternary (dead end) complex with CoA and carnitine, as well as the structure of the S554A/M564G double mutant in a ternary (Michaelis) complex with hexanoylcarnitine and CoA. The structures define in molecular detail the Michaelis complexes for both the forward and reverse reactions of the enzyme and provide significant new insights into the catalytic mechanism of CrAT and possibly other carnitine acyltransferases as well.MATERIALS AND METHODSMutagenesis, Protein Expression, and Purification—Residues 30-626 of wild-type mouse CrAT were subcloned into the pET28a vector (Novagen) and overexpressed in Escherichia coli (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The expression construct contains an N-terminal hexahistidine tag. The S554A/M564G double mutant was created with the QuikChange kit (Stratagene) and verified by sequencing.The wild-type and mutant proteins were purified following the same protocol, with nickel-agarose, anion exchange, and gel filtration chromatography. The protein was concentrated to 32 mg/ml in a solution containing 20 mm Tris (pH 8.5) and 200 mm NaCl, flash-frozen in liquid nitrogen in the presence of 5% (v/v) glycerol, and stored at -80 °C.Protein Crystallization—Crystals of wild-type CrAT in complex with carnitine and acetyl-CoA were obtained at 4 °C by the sitting drop vapor diffusion method. The reservoir solution contained 100 mm Tris (pH 8.5), 100 mm NaCl, 14% (w/v) PEG 3350, and 2.3 mm carnitine, and the protein was at 17 mg/ml concentration. The crystals were soaked in a solution containing 100 mm Bis-Tris (pH 6.5), 150 mm NaCl, 20% (w/v) PEG 3350, 2.5 mm carnitine, 5 mm acetyl-CoA, and 25% (v/v) ethylene glycol for 3 min before being flash-frozen in liquid nitrogen for data collection at 100 K. They belong to space group C2, with cell parameters of a = 163.9 Å, b = 89.2 Å, c = 122.6 Å, and β = 128.9°. There are two CrAT molecules in the crystallographic asymmetric unit. Using this soaking protocol, crystals of wild-type CrAT in complex with carnitine and CoA have also been obtained, the acetyl group being hydrolyzed during the soaking experiment.Crystals of the S554A/M564G mutant in complex with hexanoylcarnitine and CoA were obtained at 4 °C by the sitting drop vapor diffusion method. The reservoir solution contained 100 mm Tris (pH 8.5), 18% (w/v) PEG 3350, and 2.3 mm carnitine, and the protein was at 10 mg/ml concentration. The crystals were soaked in a solution containing 100 mm Bis-Tris (pH 6.5), 150 mm NaCl, 18% (w/v) PEG 3350, 2.5 mm carnitine, 5 mm hexanoyl-CoA, and 25% (v/v) ethylene glycol for 3 min before being flash-frozen for data collection. They belong to space group C2 with cell parameters of a = 164.1 Å, b = 89.1 Å, c = 122.8 Å, and β = 129.0°. These mutant crystals are isomorphous to the ternary complexes of wild-type CrAT described above, with two CrAT molecules in the crystallographic asymmetric unit.Data Collection, Structure Determination, and Refinement—X-ray diffraction data were collected on an ADSC Quantum-4 CCD at the X4A beamline of the National Synchrotron Light Source. The diffraction images were processed and scaled with the HKL package (30Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38355) Google Scholar).These ternary complex crystals have cell parameters that are similar to those of the free enzyme and binary complexes of wild-type CrAT (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), with about 5 Å difference in the a axis and 3Å in the c axis. The initial structure was determined by the molecular replacement method, with the program COMO (31Jogl G. Tao X. Xu Y. Tong L. Acta Crystallogr. Sect. D. 2001; 57: 1127-1134Crossref PubMed Scopus (89) Google Scholar), using the structure of the free enzyme as the search model. The structure refinement was carried out with the program CNS (32Brunger 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. 1998; 54: 905-921Crossref PubMed Scopus (16929) Google Scholar). Manual adjustment of the atomic model against the electron density was performed with the program O (33Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13003) Google Scholar). The crystallographic information is summarized in Table 1.TABLE 1Summary of crystallographic informationWild-type enzyme and carnitine + acetyl-CoA substrateWild-type enzyme and carnitine + CoA substrateS554A/M564G enzyme and hexanoylcarnitine + CoA substrateResolution range (Å)30-2.230-1.930-2.1No. of observations195,424204,773225,110Rmerge (%)aRmerge = ∑h∑i|Ihi - 〈Ih 〉|∑h∑iIhi. The numbers in parenthesis are for the highest resolution shell.4.0 (9.8)7.4 (40.6)4.5 (10.8)I/σI24.6 (7.8)10.8 (1.9)22.5 (6.4)Redundancy3.0 (2.4)1.9 (1.8)3.0 (2.1)No. of reflections65,954101,20575,758Completeness (%)95 (80)92 (77)94 (80)R factor (%)bR = ∑h|Foh - Fch|/∑hFoh.17.0 (19.4)19.3 (31.0)17.7 (21.1)Free R factor (%)22.3 (24.9)23.5 (32.7)21.9 (25.5)Average B for protein atoms (Å2)242221Average B for ligands (Å2)222233Residues in most favored region of the Ramachandran plot (%)90.491.790.7Residue in disallowed region of Ramachandran plotIle116Ile116Ile116r.m.s. deviation in bond lengths (Å)0.0060.0060.006r.m.s. deviation in bond angles (degrees)1.11.21.1Protein Data Bank code2H3P2H3U2H3Wa Rmerge = ∑h∑i|Ihi - 〈Ih 〉|∑h∑iIhi. The numbers in parenthesis are for the highest resolution shell.b R = ∑h|Foh - Fch|/∑hFoh. Open table in a new tab Kinetic Studies—The kinetic parameters of the wild-type and mutant CrAT were determined using an end point fluorometric assay (34Hassett R.P. Crockett E.L. Anal. Biochem. 2000; 287: 176-179Crossref PubMed Scopus (16) Google Scholar). The reaction buffer contained 40 mm Hepes (pH 7.8), 1.5 mm EDTA, and 3-500 μm acetyl-CoA or hexanoyl-CoA. Each reaction contained 10-200 ng of wild-type enzyme or the M564G mutant, or 1-4 μg of the S554A or S554A/M564G mutant, in a volume of 600 μl. The reactions were initiated by the addition of carnitine (1.5 mm final concentration), allowed to progress for 10 min at room temperature, and then stopped by heat treatment at 70 °C. The free CoA product was reacted with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide (ABD-F; Molecular Probes, Inc., Eugene, OR) by incubation at 42 °C for 40 min, and the fluorescence was recorded at 535 nm (excitation wavelength 405 nm) using a 96-well plate reader (200 μl of solution/well; PerkinElmer Life Sciences). Control reactions in the absence of carnitine were carried out to remove the background. The kinetic data were fitted to the Michaelis-Menten equation (Table 2).TABLE 2Kinetic parameters of wild-type and mutant CrATkcatKmkcat/KmS−1μMM−1 S−1 × 106With acetyl-CoA as the substrateaCarnitine is present at saturating concentrations (1.5 mM). Each assay was repeated two or three times to ensure reproducibility.Wild type97.6 ± 3.121.0 ± 2.04.7 ± 0.3S554A4.0 ± 0.492.1 ± 21.60.043 ± 0.007M564G28.4 ± 1.574.6 ± 10.90.38 ± 0.04S554A/M564G1.4 ± 0.159.6 ± 7.50.024 ± 0.002H343ANAbNA, no appreciable activity with up to 50 μg of protein in the reaction.NANAH343E15.4 ± 0.832.4 ± 4.40.48 ± 0.04With hexanoyl-CoA as the substrateaCarnitine is present at saturating concentrations (1.5 mM). Each assay was repeated two or three times to ensure reproducibility.Wild type30.4 ± 0.8103.4 ± 6.70.29 ± 0.01S554A1.42 ± 0.0441.5 ± 3.10.034 ± 0.002M564G204.7 ± 8.222.4 ± 3.59.1 ± 1.2S554A/M564G8.2 ± 0.5111.8 ± 15.90.073 ± 0.007H343ANANANAH343ENANANAa Carnitine is present at saturating concentrations (1.5 mM). Each assay was repeated two or three times to ensure reproducibility.b NA, no appreciable activity with up to 50 μg of protein in the reaction. Open table in a new tab RESULTS AND DISCUSSIONStrategies to Obtain the Ternary Complexes of CrAT—Our structures of the CoA and carnitine binary complexes of wild-type CrAT defined the binding modes of these two substrates on their own (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). We have also determined the crystal structure of the F565A mutant in a ternary (dead end) complex with carnitine and CoA (17Hsiao Y.-S. Jogl G. Tong L. J. Biol. Chem. 2004; 279: 31584-31589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). However, to fully understand the catalysis by this enzyme, the structure of the ternary (Michaelis) complex with carnitine and acetyl-CoA (or acetylcarnitine and CoA) is needed. Our earlier attempts at soaking wild-type CrAT crystals with acetyl-CoA showed that the acetyl group is quickly hydrolyzed, even in the crystalline state (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). This suggested that the catalytic activity of the enzyme must be reduced before there is any possibility of capturing the structure of the ternary (Michaelis) complex.Previous studies have indicated that His343 is the general base in the catalysis by CrAT (4McGarry J.D. Brown N.F. Eur. J. Biochem. 1997; 244: 1-14Crossref PubMed Scopus (1322) Google Scholar, 5Ramsay R.R. Gandour R.D. van der Leij F.R. Biochim. Biophys. Acta. 2001; 1546: 21-43Crossref PubMed Scopus (299) Google Scholar). Crystal structures show that this side chain is held in an unusual conformation, with a hydrogen bond between the side chain Nδ2 atom and the main chain carbonyl oxygen (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). A similar conformation for the catalytic His residue is observed in the related enzymes chloramphenicol acetyltransferase (27Leslie A.G.W. Moody P.C. Shaw W.V. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4133-4137Crossref PubMed Scopus (132) Google Scholar) and dihydrolipoyl transacetylase (35Mattevi A. Obmolova G. Schulze E. Kalk K.H. Westphal A.H. de Kok A. Hol W.G.J. Science. 1992; 255: 1544-1550Crossref PubMed Scopus (227) Google Scholar), and this conformation may enhance the reactivity of the His residue as a general base.As a first attempt to reduce the activity of the enzyme, we mutated the catalytic His343 residue of CrAT to Ala or Glu. Our kinetic studies showed that the H343A mutant is inactive toward both acetyl-CoA and hexanoyl-CoA substrates, even when 50 μg of the enzyme were used in the assay (Table 2). In comparison, only 10 ng of the wild-type enzyme is needed to obtain robust activity. This suggests that the H343A mutation caused a >5,000-fold reduction in the activity of CrAT, consistent with the crucial role of this residue in catalysis. On the other hand, the H343E mutant has only a 10-fold loss in kcat/Km toward the acetyl-CoA substrate (Table 2), suggesting that the carboxylic side chain of the Glu residue can partially function as the general base for catalysis. The H343E mutant, however does not show appreciable activity toward the hexanoyl-CoA substrate (Table 2). The presence of the negative charge may disfavor substrates with longer aliphatic chains.Unfortunately, we could not observe any electron density for the CoA molecule in the active site region for either mutant (data not shown), indicating that it was mostly disordered in these mutants. Therefore, it appears that the His343 side chain may also play a role in stabilizing the conformation of the CoA molecule.Based on our structural analysis, the Ser554 residue is part of the oxyanion hole and helps to stabilize the tetrahedral transition state intermediate of the catalysis (16Jogl G. Tong L. Cell. 2003; 112: 113-122Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Earlier kinetic studies on CrOT showed that mutation of this Ser residue to Ala produced a 10-fold decrease in the kcat while having little impact on the Km for carnitine (36Cronin C.N. Biochem. Biophys. Res. Commun. 1997; 238: 784-789Crossref PubMed Scopus (18) Google Scholar). Therefore, we produced the S554A mutant of CrAT for our attempts to obtain the ternary (Michaelis) complexes. Our kinetic studies indicated a 25-fold reduction in the kcat and a 4-fold increase in the Km for acetyl-CoA of this mutant (Table 2), suggesting that the S554A mutation may have a somewhat larger impact on the catalysis by CrAT than CrOT. We also produced the S554A/M564G double mutant, since the latter mutation increases the activity of CrAT toward medium-chain fatty acids (such as hexanoyl-CoA) (17Hsiao Y.-S. Jogl G. Tong L. J. Biol. Chem. 2004; 279: 31584-31589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37Cordente A.G. Lopez-Vinas E. Vazquez M.I. Swiegers J.H. Pretorius I.S. Gomez-Puertas P. Hegardt F.G. Asins G. Serra D. J. Biol. Chem. 2004; 279: 33899-33908Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This double mutant could then allow us to determine the binding modes of medium-chain acyl groups. Our kinetic data on this double mutant as well as the M564G single mutant (Table 2) are fully consistent with kinetic data reported earlier (17Hsiao Y.-S. Jogl G. Tong L. J. Biol. Chem. 2004; 279: 31584-31589Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 37Cordente A.G. Lopez-Vinas E. Vazquez M.I. Swiegers J.H. Pretorius I.S. Gomez-Puertas P. Hegardt F.G. Asins G. Serra D. J. Biol. Chem. 2004; 279: 33899-33908Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar).Whereas mutagenesis is a powerful way to reduce the catalytic activity of CrAT, there could be concerns in the resulting structures as to the impact of the mutations. Therefore, we also developed a soaking protocol that would allow us to capture the ternary (Michaelis) complex for the wild-type enzyme by sampling the pH of the soaking solution and the soaking time. The optimal pH of CrAT is 7.4, and most of the earlier soaking experiments were carried out at this pH. By lowering the pH of the soaking solution, we should be able to reduce the catalytic activity of the enzyme, since it can be expected that the His343 catalytic residue becomes a weaker base at lower pH, especially if it becomes protonated. We have varied the pH of the soaking solution from 5.5 to 8.5, together with using short soaking times (1-5 min), to obtain the simultaneous binding mode of carnitine and acetyl-CoA to the wild-type enzyme. It should be noted that the actual pH of the system could also change during the cryofreezing procedure. Structural comparisons with the binary complexes, which were obtained near the optimal pH of the enzyme, show that the reduction in the pH did not cause a significant change in the active site of the enzyme (see below).Structure of CrAT in a Ternary (Michaelis) Complex with Carnitine and Acetyl-CoA—The crystal structure of wild-type murine CrAT in a ternary complex with carnitine and acetyl-CoA has been determined at 2.2 Å resolution (Fig. 1). The atomic model contains residues 30-625 of the enzyme, with excellent agreement to the crystallographic data and expected geometric parameters (Table 1). The majority of the residues (90%) are in the most favored region of the Ramachandran plot, whereas residue Ile116 is the only one in the disallowed region (Table 1). This residue has clearly defined electron density and is also in the disallowed region in the structures of the free enzyme and the binary complexes.To obtain this structure, wild-type murine CrAT was first co-crystallized with carnitine and then soaked in a solution containing 5 mm acetyl-CoA at pH 6.5 for 3 min. There are two molecules of CrAT in the crystallographic asymmetric unit. Clear electron density for acetyl-CoA (Fig. 2A) and carnitine was observed in the active site of one of these molecules, whereas the other active site contained carnitine and CoA. This suggests that the hydrolysis of the acetyl-CoA substrate can still occur at the lowered pH, and the enzyme is still catalytically active in the crystalline environment. Using this protocol, we also successfully obtained the crystal structure of the S554A mutant of CrAT in a ternary complex with acetyl-CoA and carnitine (data not shown). From our experiments, it appears that pH 6.5 is the optimal soaking condition for observing the electron density of acyl groups in the active site of CrAT.FIGURE 2Structure of wild-type CrAT in a ternary complex with acetyl-CoA and carnitine. A, final 2Fo - Fc electron density map for acetyl-CoA at 2.2 Å resolution, contoured at 1σ. This image was produced with Setor (40Evans S.V. J. Mol. Graphics. 1993; 11: 134-138Crossref PubMed Scopus (1249) Google Scholar). B, stereo drawing showing the active site of CrAT in the ternary complex with acetyl-CoA (brown) and carnitine (green). The side chain of His343 is shown in red, and Ser554 is shown in black. Residues 104-117 have been omitted for clarity (except for the side chain of Tyr107). A water molecule involved in carnitine binding is shown as a sphere in red. This image was produced with Ri" @default.
• W1985134465 created "2016-06-24" @default.
• W1985134465 creator A5004904484 @default.
• W1985134465 creator A5085443546 @default.
• W1985134465 creator A5090364248 @default.
• W1985134465 date "2006-09-01" @default.
• W1985134465 modified "2023-09-27" @default.
• W1985134465 title "Crystal Structures of Murine Carnitine Acetyltransferase in Ternary Complexes with Its Substrates" @default.
• W1985134465 cites W1539796472 @default.
• W1985134465 cites W1966809709 @default.
• W1985134465 cites W1985886233 @default.
• W1985134465 cites W1988632024 @default.
• W1985134465 cites W1991022642 @default.
• W1985134465 cites W1995017064 @default.
• W1985134465 cites W1997433550 @default.
• W1985134465 cites W1999576623 @default.
• W1985134465 cites W2004992318 @default.
• W1985134465 cites W2006667778 @default.
• W1985134465 cites W2013083986 @default.
• W1985134465 cites W2015832516 @default.
• W1985134465 cites W2018118901 @default.
• W1985134465 cites W2023255657 @default.
• W1985134465 cites W2029903095 @default.
• W1985134465 cites W2030975297 @default.
• W1985134465 cites W2031846075 @default.
• W1985134465 cites W2043605177 @default.
• W1985134465 cites W2049521571 @default.
• W1985134465 cites W2049850722 @default.
• W1985134465 cites W2055675213 @default.
• W1985134465 cites W2062876845 @default.
• W1985134465 cites W2066885275 @default.
• W1985134465 cites W2079901091 @default.
• W1985134465 cites W2084526009 @default.
• W1985134465 cites W2097080554 @default.
• W1985134465 cites W2105554307 @default.
• W1985134465 cites W2106315897 @default.
• W1985134465 cites W2106457742 @default.
• W1985134465 cites W2111310829 @default.
• W1985134465 cites W2113892466 @default.
• W1985134465 cites W2122844271 @default.
• W1985134465 cites W2131256962 @default.
• W1985134465 cites W2132480200 @default.
• W1985134465 cites W2151076819 @default.
• W1985134465 cites W2162723494 @default.
• W1985134465 cites W2294703791 @default.
• W1985134465 cites W4205775889 @default.
• W1985134465 cites W4241387643 @default.
• W1985134465 cites W940111687 @default.
• W1985134465 doi "https://doi.org/10.1074/jbc.m602622200" @default.
• W1985134465 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2940834" @default.
• W1985134465 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16870616" @default.
• W1985134465 hasPublicationYear "2006" @default.
• W1985134465 type Work @default.
• W1985134465 sameAs 1985134465 @default.
• W1985134465 citedByCount "21" @default.
• W1985134465 countsByYear W19851344652013 @default.
• W1985134465 countsByYear W19851344652015 @default.
• W1985134465 countsByYear W19851344652017 @default.
• W1985134465 countsByYear W19851344652020 @default.
• W1985134465 countsByYear W19851344652021 @default.
• W1985134465 countsByYear W19851344652023 @default.
• W1985134465 crossrefType "journal-article" @default.
• W1985134465 hasAuthorship W1985134465A5004904484 @default.
• W1985134465 hasAuthorship W1985134465A5085443546 @default.
• W1985134465 hasAuthorship W1985134465A5090364248 @default.
• W1985134465 hasBestOaLocation W19851344651 @default.
• W1985134465 hasConcept C104317684 @default.
• W1985134465 hasConcept C119157956 @default.
• W1985134465 hasConcept C185592680 @default.
• W1985134465 hasConcept C199360897 @default.
• W1985134465 hasConcept C2778435403 @default.
• W1985134465 hasConcept C2779649592 @default.
• W1985134465 hasConcept C2781285689 @default.
• W1985134465 hasConcept C41008148 @default.
• W1985134465 hasConcept C55493867 @default.
• W1985134465 hasConcept C64452783 @default.
• W1985134465 hasConcept C8010536 @default.
• W1985134465 hasConceptScore W1985134465C104317684 @default.
• W1985134465 hasConceptScore W1985134465C119157956 @default.
• W1985134465 hasConceptScore W1985134465C185592680 @default.
• W1985134465 hasConceptScore W1985134465C199360897 @default.
• W1985134465 hasConceptScore W1985134465C2778435403 @default.
• W1985134465 hasConceptScore W1985134465C2779649592 @default.
• W1985134465 hasConceptScore W1985134465C2781285689 @default.
• W1985134465 hasConceptScore W1985134465C41008148 @default.
• W1985134465 hasConceptScore W1985134465C55493867 @default.
• W1985134465 hasConceptScore W1985134465C64452783 @default.
• W1985134465 hasConceptScore W1985134465C8010536 @default.
• W1985134465 hasIssue "38" @default.
• W1985134465 hasLocation W19851344651 @default.
• W1985134465 hasLocation W19851344652 @default.
• W1985134465 hasLocation W19851344653 @default.
• W1985134465 hasOpenAccess W1985134465 @default.
• W1985134465 hasPrimaryLocation W19851344651 @default.
• W1985134465 hasRelatedWork W1990364992 @default.
• W1985134465 hasRelatedWork W1990519800 @default.
• W1985134465 hasRelatedWork W2006884067 @default.
• W1985134465 hasRelatedWork W2028662668 @default.

• next