Structural Basis of the Substrate-specific Two-step Catalysis of Long Chain Fatty Acyl-CoA Synthetase Dimer* □ S

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 (cid:2) -( (cid:1) , (cid:2) -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.

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 (1)(2)(3)(4)(5)(6). In single cell organisms, long chain fatty acyl-CoA synthetase (LC-FACS) 1 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 (7). This movement results in the accumulation of fatty acyl-CoA esters, the first process of ␤-oxidation (7)(8)(9)(10). 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 (11,12), enzyme activation (13), protein acylation (14), cell signaling (15), and transcriptional regulation (16).
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) (5,6). 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 (7,17,18). All FACSs catalyze a magnesiumdependent multisubstrate reaction, resulting in the formation of fatty acyl-CoA (19,20). The reaction requires ATP, a fatty acid, and CoA with an overall reaction scheme as described in Reaction 1. fatty acid ϩ CoA ϩ ATP 3 fatty acyl-CoA ϩ PPi ϩ AMP REACTION 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 ϩ ATP 3 fatty acyl-AMP ϩ PPi fatty acyl-AMP ϩ CoA 3 fatty acyl-CoA ϩ AMP REACTIONS 2 AND 3 * This work was supported in part by the National project on protein structural and functional analysis funded by Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure.
The atomic coordinates and structure factors ( The esterification of fatty acids by LC-FACS has been proposed to proceed via a Bi Uni Uni Bi Ping-Pong mechanism (21) based on extensive kinetic studies of the rat enzyme (22). 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 (23,24) in contrast to the acetyl-AMP or butyryl-AMP for SC-or MC-FACSs (25)(26)(27).
The crystal structures of four adenylate forming enzymes have so far been reported: luciferase (28), phenylalanine-activating A domain (PheA) (29), 2,3-dihydroxybenzoate-activating E domain (DhbE) (30), and acetyl CoA synthetase (SC-FACS) (31,32). 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 (29), DhbE (30) and SC-FACS (31,32). 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 (30). 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 (31,32). 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 (8,10). However, the structurefunction 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) (33) 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.

MATERIALS AND METHODS
Expression and Purification-The ttLC-FACS gene was amplified by PCR using the primers 5Ј-ATATCATATGGAAGGGGAAAGGAT-GAACGCGTTCCCAA-3Ј and 5Ј-ATATAGATCTTTATTAGGCGCCTC-CGTAGTAGTTCTTGTAC-3Ј 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 li-gated 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 g/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 M 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 (34). 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 MgCl 2 , 1 mM CoA, 0.2 mM potassium phosphoenolpyruvate, 0.15 mM NADH, 20 g/ml adenylate kinase, 30 g/ml pyruvate kinase, 30 g/ml lactate dehydrogenase, and 5 g/ml ttLC-FACS. The magnesium-free assay was performed in the presence of 10 mM EDTA instead of MgCl 2 .
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 l of ttLC-FACS solution (9. X-ray Data Collection and Processing-The diffraction data were collected using beam lines (BL26B1, BL41XU, and BL45XU) (35) 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 (36).
Structure Determination and Refinement-Initial phases were calculated by the program SOLVE (37) 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 RE-SOLVE (38), and initial model building was performed by the program ARP/wARP (39). 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 (40) and Molrep (41) 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 (42), CNS (43), and REFMAC5 (44). The dictionaries of AMP-PNP and myristoyl-AMP used for the restrained crystallographic refinement were prepared by QUANTA/CHARMm (Accelrys) and REFMAC5 (44). Although the reflections for R free 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 (43). 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 R free values of 0.25, 0.24, and 0.24, respectively. The geometrical quality was checked using PROCHECK (45). The current atomic coordinates have been deposited to the Protein Data Bank (46) with accession codes 1ULT, 1V25, and 1V26. The statistics of data collection and refinement on ttLC-FACS are summarized in Tables I and II.
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 (47) using PSI-BLAST (48) and T-coffee (49). After the sequence alignment using T-coffee (49), phylogenetic analysis was performed using PHYLIP (50). The amino acid sequence of four structurally characterized family members luciferase (28), PheA (29), DhbE (30), and SC-FACS (31) were aligned structurally with ttLC-FACS using QUANTA (Accerylys) with manual modification.

RESULTS
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 Mg 2ϩ ions (3,19,22,27) (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 (Thr 184 -Thr-Gly-Thr-Thr-Gly-Leu-Pro-Lys 192 ), the phosphate-binding site ( Fig. 2A) (7,8,10,51). The motifs were designated based on the ttLC-FACS structures presented in this paper. The L motif (Asp 432 -Arg-Leu-Lys-Asp-Leu 437 ) contains the peptide that acts as a linker between the N-and C-terminal domains, the A motif (Gly 323 -Tyr-Gly-Lue-Thr-Glu-Thr 329 ) contains the adenine-binding residue Tyr 324 , whereas the G motif (Val 226 -Pro-Met-Phe-His-Val-Asn-Ala-Trp 234 ) contains the gate residue Trp 234 and the surrounding mobile peptide as described below.
where the free reflections (5% for Apo and 10% for liganded in the total used) were held aside for R free throughout refinement. Overall Structure-ttLC-FACS forms a domain swapped dimer (52). The monomers of the dimer interact at their Nterminal domains with a contact surface area of 3600 Å 2 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 In the dimer interactions, Asp 15 forms an intermolecular salt bridge with Arg 176 . The main chain carbonyl group of Glu 16 forms an inter-molecular hydrogen bond with the side chain of Arg 199 , whereas Glu 175 and Arg 199 form an intermolecular salt bridge at the interface (Fig. 3C). The multisequence alignment of LC-FACS ( Fig. 2A) reveals that residues corresponding to Asp 15 , Glu 16 , Arg 176 , and Arg 199 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) (28 -32).
Each monomer of ttLC-FACS is composed of a large Nterminal 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 (28 -32). The N-terminal domain can be further divided into two subdomains: a distorted antiparallel ␤-barrel and two ␤-sheets that are flanked on both sides by ␣-helices forming an ␣␤␣␤␣ sandwich. The small C-terminal globular domain is comprised of a two-stranded ␤-sheet and a three-stranded antiparallel ␤-sheet that is surrounded by three ␣-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 the presence of ligand. In the AMP-PNP complex structure of ttLC-FACS, the C-terminal domains are in the closed conformation with direct interactions formed between the C-and N-terminal domains (Fig.  4A, panel 3). The closed conformation of the C-terminal domain is also maintained in the complex structure with myristoyl-AMP (Fig. 4A, panel 4). Both of these complex structures with AMP-PNP and myristoyl-AMP are almost the same closed conformation, and superimposition of the four N-and C-terminal domains of the AMP-PNP and myristoyl-AMP complex structures yields average root mean square deviations of 0.34 Å (486 C␣ atoms) and 0.57 Å (57 C␣ atoms), respectively. In contrast in the uncomplexed structure, the C-terminal domains show two types of open conformations (Fig. 4A, panels 1 and 2). The Fatty Acid-binding Tunnel with Open Gate upon ATP Binding-Compared with SC-FACS (31,32), LC-FACS should incorporate a larger fatty acid-binding site to accommodate bulkier long chain fatty acids. A fatty acid-binding tunnel was identified in the N-terminal domain of each monomer (Fig. 4, B and C), which extends from the concave cavity in the central valley to the ATP-binding site and is surrounded by a large ␤-sheet including ␤ 12 , ␤ 13 , ␤ 14 , ␤ 15 , and an ␣-helix cluster including ␣ g and ␣ h (Figs. 3D and 4B). In the complex structure this tunnel is composed of a large central pathway that is divided into two distinct paths, the "ATP path" and the "center path, " by the indole ring of Trp234 in the G motif (Fig. 4C). In addition, there is another pocket that branches from the central pathway named the "dead end branch. " All three paths are separated by the indole ring of Trp 234 in the uncomplexed structure.
The fatty acid-binding tunnel closed by the indole ring of Trp 234 in the uncomplexed structure opens upon AMP-PNP binding via hydrogen bond formation between ␤-phosphate and the ring nitrogen of Trp 234 (Fig. 4C). At the same time the mobile C-terminal domain adopts the closed conformation as seen in the adenylate binding structures (Fig. 4A, panel 3). The Ribbon representations of the ttLC-FACS dimer are shown (A). In the panel, the secondary structure of the C-terminal domain is colored in green. In the N-terminal domain, ␣-helix and ␤-sheet are colored in cyan and red, respectively, with the N-terminal domain-swapping peptide colored in yellow. The electrostatic potential surface map of ttLC-FACS dimer in the same orientation as the representation in A. Red represents negatively charged regions, and blue represents positively charged regions (B). Close-up view of the N-terminal peptide involved in domain swapping in the reverse orientation view to A (C). Residues with carbons colored in pink against a cyan surface of one monomer interacts with the concave surface of the other monomer colored in yellow. There are salt bridges at the domain swapping region. The monomer of ttLC-FACS with each secondary structure feature is labeled according to the scheme given in Fig. 2A (D).
indole ring of Trp 234 rotates by 29°and Ϫ81°around the angles of 1 and 2, respectively in the complexed structures compared with the uncomplexed structure (Fig. 4C). The ring nitrogen of Trp 234 , which is free in the open conformation forms a hydrogen bond with the ␤-phosphate in the AMP-PNP complex structure. Furthermore, in the closed structures of ttLC-FACS, the dead end branch is wider than that of the uncomplexed forms because of a shift in the flexible loop of the G motif, His 230 -Val-Asn-Ala-Trp-Cys-Leu 236 by 0.9 Å ( Figs. 2A  and 4C). The average temperature factors of this loop in the uncomplexed and complexed structures of ttLC-FACSs are 45.4 and 13.1 Å 2 , respectively.
Adenylate Binding-The crystal structures with the bound AMP-PNP allowed for a detailed characterization of adenylate binding in the closed conformation (Fig. 5). In the AMP-PNP complex structure, an AMP-PNP is bound to each monomer in a crevasse at the interface between the N-and C-terminal domains (Fig. 4A, panel 3). The AMP moiety is oriented by a combination of hydrophobic interactions as well as hydrogen bonds with Arg 433 and Lys 435 from the L motif, Lys 439 and Trp 444 from the C-terminal domain, and a number of residues from the N-terminal domain (Fig. 5A).
After soaking the complex crystals of AMP-PNP in the presence of myristate, the adenylate intermediate myristoyl-AMP was clearly identified as the acylation product of Reaction 2 in the crystals (Fig. 5B). The ␣-phosphate mediates interactions between the N-and C-terminal domains (Fig. 5). The observed hydrogen bond network between the ␣-phosphate and the Nterminal domain is essentially the same in both the AMP-PNP and the myristoyl-AMP complex structures. The putative acidic oxygen of the ␣-phosphate (O2A) forms hydrogen bonds with the backbone nitrogen and the side chain hydroxyl group of Thr 327 (Fig. 5C). Furthermore, O2A is coordinated to the essential magnesium ion (Mg 2ϩ ). The Mg 2ϩ is further bonded to the hydroxyl group of Thr 184 and the carboxyl oxygen of Glu 328 (Fig. 5D), and this is consistent with the mutation studies of FadD (8).
In contrast to the N-terminal domain, the hydrogen bond network between the ␣-phosphate and the C-terminal domain differs in the AMP-PNP and myristoyl-AMP complex structures that may reflect the two distinct acylation steps of Reactions 2 and 3 (Fig. 5D). The hydrogen bond between the side chain amino group of Lys 439 and the acidic oxygen (O1A) of the ␣-phosphate is only formed in the myristoyl-AMP complex, and the hydrogen bond distance between the nitrogen of the indole ring of Trp 444 and O1A of the ␣-phosphate increases from 2.8 Å in the AMP-PNP complex to 3.4 Å in the myristoyl-AMP complex. There is rotation around the C5Ј-O5Ј and the O5Ј-␣phosphorus bonds in the AMP moiety of 10°and 30°between the AMP-PNP and myristoyl-AMP complex structures, respectively (Fig. 5, C and D).
In the pyrophosphate moiety of the AMP-PNP complex structure, the ␤-phosphate interacts with both the N-and C-terminal domains (Fig. 5A). Fatty Acid Binding-In the fatty acid-binding tunnel, the ATP path is a hydrophobic channel connected to the ATPbinding site (Figs. 4 and 5). The center path is the entrance site for the fatty acid and extends from the dimer interface along ␤-strand 13 to the ATP path (Fig. 4B). In the absence of ATP, the indole ring of Trp 234 blocks the connection between the two paths as described (Fig. 4C). The center path is filled with ordered water molecules that can be seen in both the AMP-PNP and the myristoyl-AMP complex structures. These ordered water molecules connect to the bulk solvent region through the entrance of the center path. The entrance of the center path is located at the concave surface in the central valley of the dimer interface (Fig. 4B), which generates a positive electrostatic potential because of the basic residues (Lys 219 , Arg 296 , Arg 297 , Arg 321 , Lys 350 , and Lys 354 ) from each monomer (Fig. 3B). The third path, the dead end branch, extending from the fatty acid-binding tunnel to ␣-helix h (residues 235-243) is also gated by Trp 234 in the uncomplexed structure (Fig. 4C). The bottom of the dead end branch has a hydrophilic environment generated from water molecules and polar side chains (His 204 , Ser 209 , and Thr 214 ) (Fig. 5).
The aliphatic hydrocarbon chain of the myristoyl-AMP inserts into the dead end branch via the ATP path (Fig. 5, B and  C). The amino acid residues within a 4.5 Å radius of the myristoyl moiety are His 204 , Ala 208 , Thr 214 , Val 231 , Trp 234 , Cys 235 , Leu 236 , Ala 239 , Val 299 , Gly 301 , Gly 323 , Tyr 324 , Gly 325 , Leu 326 , Thr 327 , Pro 331 , Val 332 , and Gln 335 of the N-terminal domain and Lys 439 of the C-terminal domain. The aliphatic chain of the myristoyl-AMP is in an extended conformation with a kink between C9 and C10, resulting in a torsion angle of 59° (Fig. 5B). This gauche conformation results in the C10 to C14 portion of the aliphatic chain being positioned in the dead end branch (Fig. 5C).
Structural Comparison of Adenylate-forming Enzymes-A comparison was made of the closed conformations of the structurally characterized adenylate forming enzymes including DhbE, PheA, and SC-FACS as well as ttLC-FACS by the superimposition of the bound adenine rings in the complex structures (Fig. 6) (29 -32). There are two-type of closed C-terminal domain conformations of SC-FACS complexed to the ligands, AMP (32) and propyl-AMP (31); one is the DhbE (30) and PheA (29) type, and the other is the ttLC-FACS type. These two conformations show almost reverse orientations of C-terminal domain relative to the N-terminal domain. The position of the C-terminal domain differs by 180°in the two types. The protein backbones are oriented in opposite directions after the linker region, resulting in the putative catalytic residue differences. (Fig. 6) (31). DISCUSSION ATP-dependent Closure of the C-terminal Domain-Based on the crystal structures of both the uncomplexed and complexed ttLC-FACS, the ATP-dependent closure of the C-terminal domain is concluded to be the reactive conformation. This reactive conformation is stabilized by extensive noncovalent interactions involving residues conserved only among LC-FACSs. In addition this closed conformation should be maintained through the whole catalytic reaction (Figs. 4 and 5), meaning that the fatty acyl-AMP intermediate is unlikely to be released from the enzyme. This is consistent with extensive experimentation that has been unable to isolate such intermediates (23,24). In contrast to LC-FACS, SC-FACS is suggested to adopt two different closed conformations for the two steps of the reaction (31,32), whereas the catalytic Bi Uni Uni Bi Ping-Pong mechanism should be the same in all FACSs (22,(25)(26)(27). If it is the case, the conformational change via the open form between the two distinct C-terminal forms in the two-step catalysis should take place in SC-or MC-FACSs, because these FACSs were shown to release and utilize the acetyl-AMP or butyryl-AMP as the two-step reaction intermediate, respectively, but not in LC-FACS (23,24,26,27).
When the closed conformation is stabilized by the binding of ATP, ttLC-FACS is able to readily catalyze the formation of the fatty acyl-AMP intermediate using a fatty acid entering the active site that opens upon ATP binding (Reaction 2). In fact, the first half-reaction of ttLC-FACS was shown to propagate in the AMP-PNP complex crystals soaked with myristate without crystal damage, resulting in the tightly bound myristoyl-AMP The amino acid residues of the ATP-binding site are shown as stick models. The carbon atoms of the residues of the N-and Cterminal domains and the linker peptide are colored in green, magenta, and cyan, respectively, and other atoms are colored in red (oxygen), blue (nitrogen), and yellow (phosphorus). The sigmaA weighted F o Ϫ F c electron density map is 3 (silver) and 5 (orange) (57). B, the bound myristoyl-AMP in the ttLC-FACS complex structure shown is as a ball and stick model (sky blue). The sigmaA weighted F o Ϫ F c electron density map of bound myristoyl-AMP (gold) was contoured at 3. C, schematic drawing of the polar interactions in ttLC-FACS and bound myristoyl-AMP. The aliphatic chains from C1 to C9 and from C10 to C14 occupy the ATP path and the dead end branch of the fatty acidbinding tunnel, respectively. D, stereo representation of the loop structure around the substrate binding site. The loops (brown) surrounding the bound myristoyl-AMP (carbon atoms in sky blue) are shown with the putative catalytic residues (light green, carbon) and a superimposed bound AMP-PNP molecule. The bound magnesium ion is colored in pink. The regions corresponding to the LC-FACS specific conserved motifs are labeled as well as the P-loop. as the reaction intermediate (Fig. 5B). The myristoyl-AMP is the substrate for the second reaction with CoA, which takes place in the closed form without any further conformational rearrangement of the enzyme (Reaction 3). Indeed, the crystal structures of ttLC-FACS complexes with AMP-PNP and myristoyl-AMP showed the same closed conformation both before and after myristoyl-AMP formation; thus, the overall catalysis is concluded to proceed to completion, i.e. the release of fatty acyl CoA and AMP, in a fixed conformation of the C-terminal domain initiated by ATP binding.
ATP Binding-dependent Conformational Changes-Prior to the binding of a fatty acid molecule, a molecule of ATP is required to bind into ttLC-FACS, an event that results in adoption of the closed conformation and the opening of the Trp 234 gate in the fatty acid-binding tunnel (Fig. 4). In the closed conformation with bound ATP, the fatty acid-binding tunnel penetrates the N-terminal domain from the central valley of the dimer interface to the ATP-binding site connecting to the funnel cavity in both AMP-PNP and myristoyl-AMP complex structures (Figs. 4 and 5). A long chain fatty acid can enter the gate-opened fatty acid-binding tunnel through the center path from the central valley of the dimer (Fig. 4B). The aliphatic carbon tail of the myristoyl-AMP extends into the bottom of the dead end branch via the ATP path in the complex structure (Fig. 5C). In the structure of the uncomplexed enzyme, the indole ring of Trp 234 closes the junction between the ATP path and the additional paths in the fatty acid-binding tunnel. In the AMP-PNP complex structure, the Trp 234 gate is open and the dead end branch is wider than that of the uncomplexed ttLC-FACS because of a shift of the G motif loop. This shift is the result of the formation of a hydrogen bond between ␤-phosphate and the ring nitrogen of Trp 234 (Fig. 4C). The formation of a salt bridge between the side chains of His 230 in the loop and Glu 443 on the closing C-terminal domain, residues that are conserved in all except the E. coli LC-FACSs, may also be involved in the movement of the G motif (Fig. 5D).
Unidirectional Binding and Release of Fatty Acid and Its Product Fatty Acyl-CoA-In the closed conformation of the enzyme, the fatty acid should travel unidirectionally through the gate-opened fatty acid-binding tunnel from the central valley of the dimer interface to the ATP-binding site (Figs. 4 and  5). The positively charged entrance to the tunnel located at the dimer interface should attract the negatively charged carboxyl group of the long chain fatty acid (Fig. 3B). The final products, fatty acyl-CoA and AMP, should only be released from the side of the ATP-binding site after opening of the C-terminal domain. This is due to the fact that both the fatty acyl-CoA and AMP contain adenylate moieties that are too bulky to pass back through the fatty acid-binding tunnel to the central valley (Fig. 4B).
Structural Determinants of Fatty Acid Specificity-The depth of the dead end branch of the fatty acid-binding tunnel of ttLC-FACS determines the chain length and specificity of the fatty acid substrate (Figs. 4C and 5). The in vitro enzyme assay demonstrated that myristate (C14) and palmitate (C16) are good substrates at 25°C, whereas acylation of laurate (C12) and stearate (C18) proceeds but at a reduced level, which is consistent with previous results (19,20). The distance between C14 and the bound water at the bottom of the fatty acidbinding tunnel is 3.5 Å in the myristoyl-AMP complex. Palmitate fits well into the tunnel because the extra carbon atoms occupy a further length of 2.6 Å. Thus, the depth of the dead end branch accounts for substrate specificity by its compatibility with the fatty acid chain length.
The hydrophilic dead end branch may also contribute to the release of the final product, fatty acyl-CoA. In the AMP-PNP complex structure the dead end branch is lined with polar side chains and two bound water molecules. The hydrophilic environment is surrounded by the hydrophobic aliphatic chain of the bound myristoyl-AMP in the complex structure and would promote release of the fatty acyl-CoA from the fatty acid-binding tunnel enthalpically.
The diameter of the opening at the center path (3.5 Å) may be a selectivity filter for distinguishing saturated fatty acids over unsaturated ones. The unsaturated fatty acids myristoleic acid and palmitoleic acid were not substrates for ttLC-FACS under the described assay conditions at 25°C. The diameter of the tunnel for unsaturated fatty acids would need to be wider than that for the saturated fatty acid because of the rigid and bulky aliphatic chain containing the 9-cis double bond. However, it cannot be excluded that ttLC-FACS catalyzes acylation of these fatty acids to some extent at higher temperatures, closer to physiological for this enzyme, because assays were only carried out at 25°C.
Structural Basis of ttLC-FACS Acylation Catalysis-The molecular mechanism of LC-FACS is proposed to be compatible with the Bi Uni Uni Bi Ping-Pong based on the three high resolution structures of ttLC-FACS, which include the uncomplexed form and two liganded complexes (Fig. 7) (22, 53).
The reaction scheme is summarized as follows. First, the binding of ATP induces the closed conformation and opening of the Trp 234 gate of the fatty acid-binding tunnel (Fig. 7, A and  B). Second, the fatty acid molecule binds and the fatty acyl-AMP intermediate is formed (Fig. 7B). Third, the pyrophosphate molecule leaves (Fig. 7C). Fourth, a CoA molecule binds and the final product fatty acyl-CoA is formed (Fig. 7, C and D). Fifth, the fatty acyl-CoA followed by the AMP leave after opening of the C-terminal domain (Fig. 7E) (21). This scheme is fully consistent with the extended kinetic studies (22). In contrast to SC-and MC-FACS as well as other adenylate forming enzymes, the deeply buried fatty acyl-AMP intermediate is not released in the tightly closed conformation of LC-FACS (25-27, 31, 32).
For the first reaction step (Reaction 2), the short hydrogen bond between ␣-phosphorus and Trp 444 (2.8 Å) results in an electron deficient ␣-phosphorus atom to form an electrophile in the AMP-PNP complex structure (Figs. 4C and 5D). The positive charge of the bound Mg 2ϩ coordinated with O2A can additionally contribute to the electron deficiency of ␣-phosphorus. The fatty acid-binding tunnel connects to the ATP-binding site and opens to a plane formed of O5Ј, O2A, and O3A in the ␣-phosphate (Figs. 4C and 5, A and D). Thus, the carboxyl group of the fatty acid would approach the electron deficient ␣-phosphorus atom in the manner of the adjacent mechanism to form a putative pentacovalent intermediate (54). According to the adjacent mechanism, O1A in the pentacovalent intermediate should be negatively charged, which would be stabilized by the hydrogen bond between O1A and the indole ring of Trp 444 in the first reaction step (Figs. 4C and 5D).
Multiple hydrogen bonds formed between Lys 439 and the adenylate moiety are likely to play an important role in the second step of catalysis (Reaction 3) (Fig. 5, C and D). In the bound myristoyl-AMP structure the side chain amino group of Lys 439 forms hydrogen bonds with the carbonyl oxygen atom (2.7 Å) and the O1A of the ␣-phosphate (3.3 Å), generating an electron deficient carbonyl carbon and stabilizing the negative charge on the AMP leaving group, respectively. Lys 439 and Trp 444 of the C-terminal domain are involved in both reactions in the two-step catalysis, and are conserved in yeast, rat, and human LC-FACSs but not in E. coli ( Fig. 2A).
In the multisequence alignment of LC-FACS proteins, there are three conserved motifs, the G, A, and L motifs, specific to LC-FACSs in addition to the P-loop, which is conserved among all classes of adenylate forming enzymes (Figs. 2 and 5D) (7,8). The L motif may be important as a determinant for the alternative closed conformations of the C-terminal domain in the different classes of adenylate forming proteins as described. The L motif consensus sequence for all LC-FACSs is Asp 432 -Arg-Xaa-Lys 435 ( Fig. 2A), whereas for the SC-and MC-FACSs as well as DhbE and PheA the consensus motif sequence is Gly-Arg-Xaa-Asp (Fig. 2B) (29 -32). The sequence of luciferase is close to that of LC-FACSs (Fig. 2B) (28). In LC-FACSs a linker missing the Gly residue should be less flexible compared with SC-and MC-FACSs. This extra flexibility conferred by the Gly residue may explain why SC-and MC-FACSs adopt two different closed conformations of the C-terminal domain with the bound AMP (32) and propyl-AMP (31), respectively (Fig. 6). This mobility of the C-terminal domain in SC-and MC-FACSs should allow the release and utilization of the acyl adenylate intermediate (25)(26)(27). During the final stages of preparation of this manuscript, new data has been published on a group of acyl-AMP ligases from Mycobacterium tuberculosis that release acyl-AMP as product. In these enzymes there is a conserved Gly residue that is the first residue of the L motif as in SC-and MC-FACSs. This is consistent with the role of the L motif (55).
In ttLC-FACS the G motif contains the gating residue Trp 234 . Phe 247 in the G motif of firefly luciferase may be the gating residue of luciferase (Fig. 2B), because luciferase possesses LC-FACS catalytic activity (56), and the Phe is equivalent to Trp 234 of ttLC-FACS structurally. Based on the conservation of these motifs, luciferase is anticipated to evolve from an ancestral LC-FACS keeping its LC-FACS activity (56), whereas DhbE (30) and PheA (29) may have evolved from of SC-FACS. Thus, ttLC-FACS may be an archetype both of eubacterial and eukaryotic LC-FACSs, and the proposed acylation of fatty acid by ttLC-FACS may be a model for the ATP-FIG. 7. The schematic mechanism for the unidirectional ordered catalysis by ttLC-FACS. In the proposed overall catalysis of the acylation, the substrate fatty acid is processed, unidirectionally through the fatty acid-binding tunnel in the N-terminal domain and funnel cavity at the interface of N-and Cterminal domains from the central valley of the dimer interface. This schematic is based on Fig. 4B. The binding of ATP to ttLC-FACS is the initial event in the catalysis process (A); it is the trigger for both closing the C-terminal domain and the opening and widening of the gated fatty acid-binding tunnel (B). The fatty acid-binding tunnel conveys the substrate molecule unidirectionally from the positively charged concave in the central valley of the dimer interface to the ATPbinding site (B). The pyrophosphate is released after the formation of a fatty acyl-AMP. A CoA then binds to the fatty acyl-AMP complex (C). The thiol group of the bound CoA attacks the acyl carbon of the fatty acyl-AMP (D). Opening the Nand C-terminal domains again, the fatty acyl-CoA and AMP products are released from the ttLC-FACS (E). Overall catalysis is represented after Cleland's expression (F) (21). dependent vectorial transport of fatty acid (Fig. 7). The precise biological function of ttLC-FACS remains to be clarified under physiological conditions.