Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m400100200
ISSN1083-351X
AutoresYuko Hisanaga, Hideo Ago, Noriko Nakagawa, Keisuke Hamada, K. Ida, Masaki Yamamoto, Tetsuya Hori, Yasuhiro Arii, Mitsuaki Sugahara, Seiki Kuramitsu, Shigeyuki Yokoyama, Masashi Miyano,
Tópico(s)Protein Structure and Dynamics
ResumoLong chain fatty acyl-CoA synthetases are responsible for fatty acid degradation as well as physiological regulation of cellular functions via the production of long chain fatty acyl-CoA esters. We report the first crystal structures of long chain fatty acyl-CoA synthetase homodimer (LC-FACS) from Thermus thermophilus HB8 (ttLC-FACS), including complexes with the ATP analogue adenosine 5′-(β,γ-imido) triphosphate (AMP-PNP) and myristoyl-AMP. ttLC-FACS is a member of the adenylate forming enzyme superfamily that catalyzes the ATP-dependent acylation of fatty acid in a two-step reaction. The first reaction step was shown to propagate in AMP-PNP complex crystals soaked with myristate solution. Myristoyl-AMP was identified as the intermediate. The AMP-PNP and the myristoyl-AMP complex structures show an identical closed conformation of the small C-terminal domains, whereas the uncomplexed form shows a variety of open conformations. Upon ATP binding, the fatty acid-binding tunnel gated by an aromatic residue opens to the ATP-binding site. The gated fatty acid-binding tunnel appears only to allow one-way movement of the fatty acid during overall catalysis. The protein incorporates a hydrophobic branch from the fatty acid-binding tunnel that is responsible for substrate specificity. Based on these high resolution crystal structures, we propose a unidirectional Bi Uni Uni Bi Ping-Pong mechanism for the two-step acylation by ttLC-FACS. Long chain fatty acyl-CoA synthetases are responsible for fatty acid degradation as well as physiological regulation of cellular functions via the production of long chain fatty acyl-CoA esters. We report the first crystal structures of long chain fatty acyl-CoA synthetase homodimer (LC-FACS) from Thermus thermophilus HB8 (ttLC-FACS), including complexes with the ATP analogue adenosine 5′-(β,γ-imido) triphosphate (AMP-PNP) and myristoyl-AMP. ttLC-FACS is a member of the adenylate forming enzyme superfamily that catalyzes the ATP-dependent acylation of fatty acid in a two-step reaction. The first reaction step was shown to propagate in AMP-PNP complex crystals soaked with myristate solution. Myristoyl-AMP was identified as the intermediate. The AMP-PNP and the myristoyl-AMP complex structures show an identical closed conformation of the small C-terminal domains, whereas the uncomplexed form shows a variety of open conformations. Upon ATP binding, the fatty acid-binding tunnel gated by an aromatic residue opens to the ATP-binding site. The gated fatty acid-binding tunnel appears only to allow one-way movement of the fatty acid during overall catalysis. The protein incorporates a hydrophobic branch from the fatty acid-binding tunnel that is responsible for substrate specificity. Based on these high resolution crystal structures, we propose a unidirectional Bi Uni Uni Bi Ping-Pong mechanism for the two-step acylation by ttLC-FACS. Long chain fatty acyl-CoA synthetases participate in the first reaction step of long chain fatty acid degradation in various organisms from bacteria to mammals and including plants (1O'Brien W.J. Frerman F.E. J. Bacteriol. 1977; 132: 532-540Google Scholar, 2Mishina M. Kamiryo T. Tashiro S. Hagihara T. Tanaka A. Fukui S. Osumi M. Numa S. Eur. J. Biochem. 1978; 89: 321-328Google Scholar, 3Krisans S.K. Mortensen R.M. Lazarow P.B. J. Biol. Chem. 1980; 255: 9599-9607Google Scholar, 4Fulda M. Shockey J. Werber M. Wolter F.P. Heinz E. Plant J. 2002; 32: 93-103Google Scholar, 5Schomburg D. Schomburg I. 2nd Ed. Springer Handbook of Enzymes. 2. Springer-Verlag, Berlin2001: 186-218Google Scholar, 6Schomburg I. Chang A. Ebeling C. Gremse M. Heldt C. Huhn G. Schomburg D. Nucleic Acids Res. 2004; 32: D431-D433Google Scholar). In single cell organisms, long chain fatty acyl-CoA synthetase (LC-FACS) 1The abbreviations used are: LC-FACS, long chain fatty acyl-CoA synthetase; ttLC-FACS, long chain fatty acyl CoA synthetase from T. thermophilus HB8; AMP-PNP, adenosine 5′-(β,γ-imido) triphosphate; FACS, fatty acyl-CoA synthetase; SC, short chain; MC, medium chain; LC, long chain; PheA, phenylalanine-activating A domain; DhbE, 2,3-dihydroxybenzoate-activating E domain; MES, 4-morpholineethanesulfonic acid; L motif, linker motif; A motif, adenine motif; G motif, gate motif. also participates in the transport of various xenobiotic fatty acids. The LC-FACSs in Escherichia coli and Saccharomyces cerevisiae, FadD and Faa1p/Faa4p, are involved in the vectorial movement of exogenous fatty acids across the plasma membrane together with the respective fatty acid transport proteins, FadL and Fat1p (7Black P.N. DiRusso C.C. Microbiol. Mol. Biol. Rev. 2003; 67: 454-472Google Scholar). This movement results in the accumulation of fatty acyl-CoA esters, the first process of β-oxidation (7Black P.N. DiRusso C.C. Microbiol. Mol. Biol. Rev. 2003; 67: 454-472Google Scholar, 8Weimar J.D. DiRusso C.C. Delio R. Black P.N. J. Biol. Chem. 2002; 277: 29369-29376Google Scholar, 9Zou Z. Tong F. Faergeman N. Beargeman C. Black P.N. DiRusso C. J. Biol. Chem. 2003; 278: 16414-16422Google Scholar, 10Black P.N. DiRusso C.C. Sherin D. MacColl R. Kundsen J. Weimar J.D. J. Biol. Chem. 2000; 275: 38547-38553Google Scholar). In mammals LC-FACS is involved in the physiological regulation of various cellular functions through the production of long chain fatty acyl-CoA esters, which have been reported to affect protein transport (11Glick B.S. Rothman J.E. Nature. 1987; 326: 309-312Google Scholar, 12Pfanner N. Glick B.S. Arden S.R. Rothman J.E. J. Cell Biol. 1990; 110: 955-961Google Scholar), enzyme activation (13Lai J.C. Liang B.B. Jarvi E.J. Cooper A.J. Lu D.R. Res. Commun. Chem. Pathol. Pharmacol. 1993; 82: 331-338Google Scholar), protein acylation (14Li Z.N. Hongo S. Sugawara K. Sugahara K. Tsuchiya E. Matsuzaki Y. Nakamura K. J. Gen. Virol. 2001; 82: 1085-1093Google Scholar), cell signaling (15Murakami K. Ide T. Nakazawa T. Okazaki T. Mochizuki T. Kadowaki T. Biochem. J. 2001; 353: 231-238Google Scholar), and transcriptional regulation (16van Aalten D.M. DiRusso C.C. Knudsen J. EMBO J. 2001; 20: 2041-2050Google Scholar). Three types of FACS have been defined with respect to the length of the aliphatic chain of the substrate: short, medium, and long chain fatty acyl-CoA synthetases (SC-, MC-, and LC-FACSs; EC 6.2.1.1, EC 6.2.1.2, and EC 6.2.1.3, respectively) (5Schomburg D. Schomburg I. 2nd Ed. Springer Handbook of Enzymes. 2. Springer-Verlag, Berlin2001: 186-218Google Scholar, 6Schomburg I. Chang A. Ebeling C. Gremse M. Heldt C. Huhn G. Schomburg D. Nucleic Acids Res. 2004; 32: D431-D433Google Scholar). These utilize C2-C4, C4-C12, and C12-C22 fatty acids as substrates, respectively. Recent studies report that Fat1p and its homologues possess very long chain fatty acyl-CoA synthetase activity and belong to the superfamily of the adenylate forming enzymes (7Black P.N. DiRusso C.C. Microbiol. Mol. Biol. Rev. 2003; 67: 454-472Google Scholar, 17Watkins P.A. Lu J.-F. Steinberg S.J. Gould S.J. Smith K.D. Braiterman L.T. J. Biol. Chem. 1998; 273: 18210-18219Google Scholar, 18Fraisl P. Forss-Petter S. Zigman M. Berger J. Biochem. J. 2004; 377: 85-93Google Scholar). All FACSs catalyze a magnesium-dependent multisubstrate reaction, resulting in the formation of fatty acyl-CoA (19Saunders C. Voigt J.M. Weis M.T. Biochem. J. 1996; 313: 849-853Google Scholar, 20Bar-Tana J. Rose G. Shapiro B. Biochem. J. 1971; 122: 353-362Google Scholar). The reaction requires ATP, a fatty acid, and CoA with an overall reaction scheme as described in Reaction 1. fatty acid+CoA+ATP→fatty acyl-CoA+PPi+AMP REACTION 1Reaction 1 The FACS family catalyzes the formation of fatty acyl-CoA in two discrete steps: 1) the formation of a fatty acyl-AMP molecule as a stable intermediate (Reaction 2) and 2) the formation of a fatty acyl-CoA molecule as the final product (Reaction 3). fatty acid+ATP→fatty acyl-AMP+PPifatty acyl-AMP+CoA→fatty acyl-CoA+AMP REACTIONS 2 AND 3Reaction 2 The esterification of fatty acids by LC-FACS has been proposed to proceed via a Bi Uni Uni Bi Ping-Pong mechanism (21Cleland W.W. Biochim. Biophys. Acta. 1963; 67: 104-137Google Scholar) based on extensive kinetic studies of the rat enzyme (22Bar-Tana J. Rose G. Brandes R. Shapiro B. Biochem. J. 1973; 131: 199-209Google Scholar). However, to date the fatty acyl-AMP intermediate has not been isolated nor utilized experimentally as substrate for the second step (Reaction 3) or as product for the first step (Reaction 2) in reverse catalysis (23Bar-Tana J. Rose G. Shapiro B. Biochem. J. 1973; 135: 411-416Google Scholar, 24Philipp D.P. Parsons P. J. Biol. Chem. 1979; 254: 10785-10790Google Scholar) in contrast to the acetyl-AMP or butyryl-AMP for SC- or MC-FACSs (25Berg P. J. Biol. Chem. 1956; 222: 991-1013Google Scholar, 26Guranowski A. Gunther Sillero M.A. Sillero A. J. Bacteriol. 1994; 176: 2986-2990Google Scholar, 27Takao S. Ito T. Tamida M. Agric. Biol. Chem. 1987; 51: 145-152Google Scholar). The crystal structures of four adenylate forming enzymes have so far been reported: luciferase (28Conti E. Franks N.P. Brick P. Structure. 1996; 4: 287-298Google Scholar), phenylalanine-activating A domain (PheA) (29Conti E. Stachelhaus T. Marahiel M.A. Brick P. EMBO J. 1997; 16: 4174-4183Google Scholar), 2,3-dihydroxybenzoate-activating E domain (DhbE) (30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar), and acetyl CoA synthetase (SC-FACS) (31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar, 32Jogl G. Tong L. Biochemistry. 2004; 43: 1425-1431Google Scholar). The members of this superfamily show a 20–30% amino acid sequence identity with several highly conserved regions, and all catalyze the formation of acyl-AMP from ATP and a carboxylated molecule including a fatty acid and an amino acid. All four enzymes consist of a large N-terminal and a small C-terminal domain, with the catalytic site formed at the junction between the two domains. The relative positions of the C- and N-terminal domains may change upon substrate binding. In the absence of substrate, the C-terminal domain of luciferase was shown to be in an open conformation. Upon substrate binding, a closed conformation is adopted where the C- and N-terminal domains approach one another, thus reducing the accessibility of the active site to solvent as reported in the crystal structures of substrate-bound PheA (29Conti E. Stachelhaus T. Marahiel M.A. Brick P. EMBO J. 1997; 16: 4174-4183Google Scholar), DhbE (30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar) and SC-FACS (31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar, 32Jogl G. Tong L. Biochemistry. 2004; 43: 1425-1431Google Scholar). However, there does appear to be a certain amount of variability in the extent of these conformational changes because the uncomplexed form of DhbE showed a closed conformation (30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar). Furthermore the structure of SC-FACS with bound substrate analogue for the second half-reaction revealed the existence of the reverse closed conformation of the C-terminal domain compared with those of PheA and DhbE, suggesting that another large structural rearrangement is needed between the first and second reactions as supported by the recent crystal studies of SC-FACS with AMP (31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar, 32Jogl G. Tong L. Biochemistry. 2004; 43: 1425-1431Google Scholar). LC-FACS has been presumed to catalyze acylation in the same manner because of the sequence similarity and the results of extensive mutation studies based on homology modeling using the known crystal structures (8Weimar J.D. DiRusso C.C. Delio R. Black P.N. J. Biol. Chem. 2002; 277: 29369-29376Google Scholar, 10Black P.N. DiRusso C.C. Sherin D. MacColl R. Kundsen J. Weimar J.D. J. Biol. Chem. 2000; 275: 38547-38553Google Scholar). However, the structure-function relationship of LC-FACS was still unclear particularly with respect to the formation and subsequent processing of the acyl-AMP intermediate. We have determined the first three-dimensional structure of a LC-FACS domain swap homodimer from an extreme thermophile, Thermus thermophilus HB8 (ttLC-FACS) (33Yokoyama S. Hirota H. Kigawa T. Yabuki T. Shirouzu M. Terada T. Ito Y. Matsuo Y. Kuroda Y. Nishimura Y. Kyogoku Y. Miki K. Masui R. Kuramitsu S. Nat. Struct. Biol. 2000; 7: 943-945Google Scholar) overexpressed in E. coli. We also determined the structure of the enzyme complexed to AMP-PNP, a nonhydrolyzable ATP analogue. Furthermore, we identified the acyl adenylate intermediate as myristoyl-AMP in the complex crystal structure using the crystals of the AMP-PNP complex acylated by soaking in myristate solution. Based on these high resolution structures, we propose a two-step catalytic mechanism for ttLC-FACS involving a single closed conformation induced by ATP binding (Reaction 2). ttLC-FACS possesses a gated fatty acid-binding tunnel with a dead end branch in each monomer. The unidirectional movement of fatty acid is proposed as a unidirectional Bi Uni Uni Bi Ping-Pong mechanism based on these crystal structures. Expression and Purification—The ttLC-FACS gene was amplified by PCR using the primers 5′-ATATCATATGGAAGGGGAAAGGATGAACGCGTTCCCAA-3′ and 5′-ATATAGATCTTTATTAGGCGCCTCCGTAGTAGTTCTTGTAC-3′ from T. thermophilus HB8 cDNA. The amplified gene fragment was cloned into the pT7Blue (Novagen). After confirmation of the nucleotide sequence, the ttLC-FACS gene was ligated into the expression vector pET-11a (Novagen) at the NdeI/BamHI sites. The ttLC-FACS expression plasmid was transformed into E. coli strain BL21 (DE3) (Novagen) for overexpression. The cells were cultured at 37 °C in the presence of 100 μg/ml of ampicillin in LB medium for 20 h and harvested by centrifugation. The centrifuged pellet was resuspended in 20 mm Tris-HCl (pH 8.0) and heated to 70 °C for 11.5 min. All subsequent steps were performed at 4 °C. After centrifugation, ammonium sulfate was added to the supernatant, and the 30–60% (w/v) fraction was applied sequentially to HiTrap-Q HP and HiTrap-Blue HP columns (Amersham Biosciences) in the presence of buffer containing 20 mm Tris-HCl (pH 8.0), 1 mm EDTA, 1 mm dithiothreitol, and 10 μm phenylmethanesulfonyl fluoride. The purity of samples was verified using SDS-PAGE stained by Coomassie Brilliant Blue, which confirmed the presence of a single band of about 60 kDa eluted from the HiTrap-Blue HP chromatography (supplemental figure). Assay of ttLC-FACS Activity—The activity of acyl-CoA synthetase was assayed at 25 °C by an enzyme coupled spectrophotometric method (34Hosaka K. Mishina M. Tanaka T. Kamiryo T. Numa S. Eur. J. Biochem. 1979; 93: 197-203Google Scholar). The assay measures the rate of AMP formation by coupling the reaction of acyl-CoA synthetase with those of adenylate kinase, pyruvate kinase, and lactase dehydrogenase and then detects the oxidation of NADH at 334 nm with a spectrophotometer (SHIMAZU UV-2100PC). The standard reaction mixture for this assay contains 0.1 m Tris-HCl (pH 7.4), 5 mm dithiothreitol, 1.6 mm Triton X-100, 7.5 mm ATP, 10 mm MgCl2, 1 mm CoA, 0.2 mm potassium phosphoenolpyruvate, 0.15 mm NADH, 20 μg/ml adenylate kinase, 30 μg/ml pyruvate kinase, 30 μg/ml lactate dehydrogenase, and 5 μg/ml ttLC-FACS. The magnesium-free assay was performed in the presence of 10 mm EDTA instead of MgCl2. Crystallization—The crystals of the uncomplexed ttLC-FACS were obtained by the hanging drop vapor diffusion method at 20 °C. The crystallization drops were prepared by mixing 3 μl of ttLC-FACS solution (9.2 mg/ml purified ttLC-FACS, 20 mm Tris-HCl, pH 8.0, 10 μm phenylmethanesulfonyl fluoride) with 3 μl of reservoir solution (0.1 m sodium citrate, pH 5.5, 0.2 m ammonium sulfate, 21% (w/v) polyethylene glycol 4000, and 0.1 m guanidine hydrochloride). The crystals grew to dimensions 0.3 × 0.15 × 0.1 mm within 1 month. The substitution of 10 mm CoA for guanidine hydrochloride improved both the reproducibility and quality of the crystal. These improved crystals were used to prepare heavy atom derivatives by soaking with a solution containing 21% (w/v) polyethylene glycol 4000, 0.1 m sodium citrate (pH 5.5), 0.2 m ammonium sulfate, 10 mm CoA, and 1 mm thimerosal for 5 days. Crystals of the AMP-PNP complex were obtained by hanging drop vapor diffusion at 20 °C by mixing 3 μl of ttLC-FACS solution (14 mg/ml ttLC-FACS, 10 mm AMP-PNP, 10 mm CoA, 20 mm Tris-HCl, pH 8.0, 10 μm phenylmethanesulfonyl fluoride) and 3 μl of reservoir solution (50 mm MES/NaOH, pH 6.5, 0.1 m ammonium sulfate, 15% (w/v) polyethylene glycol mono methyl ester 5000). The myristoyl-AMP complex crystals were prepared by soaking AMP-PNP complex crystals into a sodium myristate solution (125 μm) containing 10 mm MgCl2 and 10 mm AMP-PNP for 24 h. X-ray Data Collection and Processing—The diffraction data were collected using beam lines (BL26B1, BL41XU, and BL45XU) (35Kumasaka T. Yamamoto M. Yamashita E. Moriyama H. Ueki T. Structure. 2002; 10: 1205-1210Google Scholar) at SPring-8 at 100 K. Before flash cooling, the crystals were washed with the reservoir solution containing 20% (v/v) glycerol to avoid formation of ice. The mercury derivative, thimerosal crystal was measured at four different wavelengths to obtain a data set for the multiwavelength anomalous diffraction method. All of the images were processed by HKL2000 (36Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326PubMed Google Scholar). Structure Determination and Refinement—Initial phases were calculated by the program SOLVE (37Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Google Scholar) using multiwavelength anomalous diffraction data from the mercury derivative up to 2.0 Å resolution (Tables I and II). These phases were improved by the program RESOLVE (38Terwilliger T.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 965-972Google Scholar), and initial model building was performed by the program ARP/wARP (39Morris R.J. Perrakis A. Lamzin V.S. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 968-975Google Scholar). The crystal structures of the uncomplexed ttLC-FACS, the AMP-PNP complex, and the myristoyl-AMP complex were determined by the molecular replacement method using AMoRe (40Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Google Scholar) and Molrep (41Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Google Scholar) and the atomic coordinates of the mercury derivative ttLC-FACS. Manual model building and the subsequent iterative crystallographic refinement were performed using the programs O (42Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Google Scholar), CNS (43Brünger 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: 905-921Google Scholar), and REFMAC5 (44Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar). The dictionaries of AMP-PNP and myristoyl-AMP used for the restrained crystallographic refinement were prepared by QUANTA/CHARMm (Accelrys) and REFMAC5 (44Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Google Scholar). Although the reflections for Rfree validation (5% for apo data set and 10% for ligand complexes) were independently selected for each diffraction data set, the molecular replacement method and the simulated annealing method starting from over 3000 K were performed to remove the model bias at the initial refinement cycle for each structure (43Brünger 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: 905-921Google Scholar). The crystal structures of apo ttLC-FACS at 2.55 Å, AMP-PNP complex at 2.3 Å, and myristoyl-AMP complex at 2.5 Å were refined with Rfree values of 0.25, 0.24, and 0.24, respectively. The geometrical quality was checked using PROCHECK (45Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Google Scholar). The current atomic coordinates have been deposited to the Protein Data Bank (46Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalow I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Google Scholar) with accession codes 1ULT, 1V25, and 1V26. The statistics of data collection and refinement on ttLC-FACS are summarized in Tables I and II.Table IStatistics of diffraction data The beam line/detector was BL26B1/Jupiter 210; the space group was P212121; the cell dimensions were a = 55.7 Å, b = 125.3 Å, and c = 211.0 Å; and the resolution was 50.0–2.0 Å.Mercury peakMercury edgeMercury remote 1Mercury remote 2Wavelength (Å)1.00811.00881.00001.0126Number of reflectionsMeasured547,659546,426560,590524,024Unique95,61795,54596,11595,275Completeness (all/highest shell; %)95.0/88.395.0/88.195.5/89.294.5/86.9I/σ(I) (all/highest shell)10.8/7.710.7/7.79.8/7.39.8/7.3Rmerge (all/highest shell; %)6.9/30.76.8/30.87.1/33.26.7/29.9 Open table in a new tab Table IIStatistics of refinementApoAMP-PNPmyristoyl-AMPData setBeam line/detectorBL41XU/MarCCDBL45XU/RAXIS-VBL45XU/RAXIS-VWavelength (Å)0.70900.98401.0000Resolution (Å)50-2.250-2.350-2.5Space groupP212121P212121P212121Cell dimensions (Å)a = 56.0, b = 124.7, c = 212.5a = 64.5, b = 101.2, c = 176.5a = 64.5, b = 101.4, c = 176.9No. of reflectionsMeasured300,383223,397299,868Unique48,37451,68841,062Completeness (all/highest shell; %)97.3/90.799.3/96.299.8/97.5I/σ(I) (all/highest shell)18.5/9.811.3/4.89.7/7.5Rmerge (all/highest shells; %)aRmerge = Σ |I - 〈I〉 |ΣI3.6/14.410.2/30.312.0/32.3RefinementResolution (Å)50-2.5548.8-2.346.1-2.5R (all/highest shell)bR and Rfree = Σ |Fo - Fc| / Σ Fo, where the free reflections (5% for Apo and 10% for liganded in the total used) were held aside for Rfree throughout refinement0.21/0.280.19/0.200.18/0.18Rfree (all/highest shell)bR and Rfree = Σ |Fo - Fc| / Σ Fo, where the free reflections (5% for Apo and 10% for liganded in the total used) were held aside for Rfree throughout refinement0.25/0.310.24/0.260.24/0.29Root mean square deviationIn bond distances (Å)0.0060.0090.010In bond angles (°)1.221.431.33a Rmerge = Σ |I - 〈I〉 |ΣIb R and Rfree = Σ |Fo - Fc| / Σ Fo, where the free reflections (5% for Apo and 10% for liganded in the total used) were held aside for Rfree throughout refinement Open table in a new tab Amino Acid Sequence Analysis—The amino acid sequence of ttLC-FACS was aligned with other LC-FACSs and members of other adenylate forming families in the Swiss-Prot data base (47Boeckmann B. Bairoch A. Apweiler R. Blatter M.C. Estreicher A. Gasteiger E. Martin M.J. Michoud K. O'Donovan C. Phan I. Pilbout S. Schneider M. Nucleic Acids Res. 2003; 31: 365-370Google Scholar) using PSI-BLAST (48Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Google Scholar) and T-coffee (49Notredame C. Higgins D. Heringa J. J. Mol. Biol. 2000; 302: 205-217Google Scholar). After the sequence alignment using T-coffee (49Notredame C. Higgins D. Heringa J. J. Mol. Biol. 2000; 302: 205-217Google Scholar), phylogenetic analysis was performed using PHYLIP (50Felsenstein J. Cladistics. 1989; 5: 164-166Google Scholar). The amino acid sequence of four structurally characterized family members luciferase (28Conti E. Franks N.P. Brick P. Structure. 1996; 4: 287-298Google Scholar), PheA (29Conti E. Stachelhaus T. Marahiel M.A. Brick P. EMBO J. 1997; 16: 4174-4183Google Scholar), DhbE (30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar), and SC-FACS (31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar) were aligned structurally with ttLC-FACS using QUANTA (Accerylys) with manual modification. Enzyme Activity and Sequence Analysis—The overexpressed and purified ttLC-FACS protein was shown to catalyze the esterification of a number of long chain fatty acids with CoA in the presence of Triton X-100. No activity was detected in the absence of detergent or Mg2+ ions (3Krisans S.K. Mortensen R.M. Lazarow P.B. J. Biol. Chem. 1980; 255: 9599-9607Google Scholar, 19Saunders C. Voigt J.M. Weis M.T. Biochem. J. 1996; 313: 849-853Google Scholar, 22Bar-Tana J. Rose G. Brandes R. Shapiro B. Biochem. J. 1973; 131: 199-209Google Scholar, 27Takao S. Ito T. Tamida M. Agric. Biol. Chem. 1987; 51: 145-152Google Scholar) (Fig. 1). Myristate (C14) is the most efficiently processed fatty acid at 25 °C, followed by palmitate (C16). The esterification of stearate (C18) and laurate (C12) was also catalyzed but at lower efficiency. In contrast, ttLC-FACS did not catalyze the esterification of the unsaturated fatty acids mysteroleic and palmitoleic acids. The amino acid sequence of ttLC-FACS was aligned with other LC-FACSs (Fig. 2A). Although the overall sequence homology is low, about 20% sequence identity or less to other LC-FACSs, there are conserved regions corresponding to the linker (L), adenine (A), and gate (G) motifs as well as the P-loop (Thr184-Thr-Gly-Thr-Thr-Gly-Leu-Pro-Lys192), the phosphate-binding site (Fig. 2A) (7Black P.N. DiRusso C.C. Microbiol. Mol. Biol. Rev. 2003; 67: 454-472Google Scholar, 8Weimar J.D. DiRusso C.C. Delio R. Black P.N. J. Biol. Chem. 2002; 277: 29369-29376Google Scholar, 10Black P.N. DiRusso C.C. Sherin D. MacColl R. Kundsen J. Weimar J.D. J. Biol. Chem. 2000; 275: 38547-38553Google Scholar, 51Saraste M. Sibbald P.R. Wittinghofer A. Trends Biochem. Sci. 1990; 15: 430-434Google Scholar). The motifs were designated based on the ttLC-FACS structures presented in this paper. The L motif (Asp432-Arg-Leu-Lys-Asp-Leu437) contains the peptide that acts as a linker between the N- and C-terminal domains, the A motif (Gly323-Tyr-Gly-Lue-Thr-Glu-Thr329) contains the adenine-binding residue Tyr324, whereas the G motif (Val226-Pro-Met-Phe-His-Val-Asn-Ala-Trp234) contains the gate residue Trp234 and the surrounding mobile peptide as described below. Overall Structure—ttLC-FACS forms a domain swapped dimer (52Liu Y. Eisenberg D. Protein Sci. 2002; 11: 1285-1299Google Scholar). The monomers of the dimer interact at their N-terminal domains with a contact surface area of 3600 Å2 for each monomer (Fig. 3). At the back of the domain swapping surface, there is a large electrostatically positive concave in the central valley of the homodimer (Fig. 3B). Each ttLC-FACS monomer swaps the eight residues from Ala8 to Glu16 into a ridge on the surface of the other N-terminal domain (Fig. 3C). In the dimer interactions, Asp15 forms an intermolecular salt bridge with Arg176. The main chain carbonyl group of Glu16 forms an inter-molecular hydrogen bond with the side chain of Arg199, whereas Glu175 and Arg199 form an intermolecular salt bridge at the interface (Fig. 3C). The multisequence alignment of LC-FACS (Fig. 2A) reveals that residues corresponding to Asp15, Glu16, Arg176, and Arg199 are highly conserved among the LC-FACS family but not conserved in the other related enzyme families (Fig 2B). Therefore, this type of domain swapped homodimer may be a characteristic feature in LC-FACS but not common in other adenylate forming enzyme families (Fig. 2) (28Conti E. Franks N.P. Brick P. Structure. 1996; 4: 287-298Google Scholar, 29Conti E. Stachelhaus T. Marahiel M.A. Brick P. EMBO J. 1997; 16: 4174-4183Google Scholar, 30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar, 31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar, 32Jogl G. Tong L. Biochemistry. 2004; 43: 1425-1431Google Scholar). Each monomer of ttLC-FACS is composed of a large N-terminal domain (residues 1–431) and a small C-terminal domain (residues 438–541) that are connected by a six-amino acid peptide linker, the L motif (residues 432–437) (Figs. 2 and 3D). The core region of the large N-terminal domain exhibits a fold similar to that seen in other adenylate-forming enzymes (28Conti E. Franks N.P. Brick P. Structure. 1996; 4: 287-298Google Scholar, 29Conti E. Stachelhaus T. Marahiel M.A. Brick P. EMBO J. 1997; 16: 4174-4183Google Scholar, 30May J.J. Kessler N. Marahiel M.A. Stubbs M.T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12120-12125Google Scholar, 31Gulick A.M. Starai V.J. Horswill A.R. Homick K.M. Escalante-Semerena J.C. Biochemistry. 2003; 42: 2866-2873Google Scholar, 32Jogl G. Tong L. Biochemistry. 2004; 43: 1425-1431Google Scholar). The N-terminal domain can be further divided into two subdomains: a distorted antiparallel β-barrel and two β-sheets that are flanked on both sides by α-helices forming an αβαβα sandwich. The small C-terminal globular domain is comprised of a two-stranded β-sheet and a three-stranded antiparallel β-sheet that is surrounded by three α-helices. Fixation of the C-terminal Domain with ATP Binding—In the ttLC-FACS structures, the C-terminal domain adopts open and closed conformations depending on
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