Crystallographic Trapping of the Glutamyl-CoA Thioester Intermediate of Family I CoA Transferases*

Coenzyme A transferases are involved in a broad range of biochemical processes in both prokaryotes and eukaryotes, and exhibit a diverse range of substrate specificities. The YdiF protein from Escherichia coli O157:H7 is an acyl-CoA transferase of unknown physiological function, and belongs to a large sequence family of CoA transferases, present in bacteria to humans, which utilize oxoacids as acceptors. In vitro measurements showed that YdiF displays enzymatic activity with short-chain acyl-CoAs. The crystal structures of YdiF and its complex with CoA, the first co-crystal structure for any Family I CoA transferase, have been determined and refined at 1.9 and 2.0 Å resolution, respectively. YdiF is organized into tetramers, with each monomer having an open α/β structure characteristic of Family I CoA transferases. Co-crystallization of YdiF with a variety of CoA thioesters in the absence of acceptor carboxylic acid resulted in trapping a covalent γ-glutamyl-CoA thioester intermediate. The CoA binds within a well defined pocket at the N- and C-terminal domain interface, but makes contact only with the C-terminal domain. The structure of the YdiF complex provides a basis for understanding the different catalytic steps in the reaction of Family I CoA transferases.

ent CoA derivatives in the absence of an acceptor co-substrate allowed us to capture the structure of the ␥-glutamyl-CoA thioester, a reaction intermediate. This structure allows us to propose roles for structurally conserved residues involved in substrate binding or catalysis. Based on the native and ␥-glutamyl-CoA thioester crystal structures, we propose a structural description for the steps in the Family I CoA-transferase catalytic cycle.

MATERIALS AND METHODS
Cloning, Expression, and Purification-The ydiF gene was amplified by PCR from E. coli O157:H7 genomic DNA (18) using Pfu polymerase (Stratagene) and oligonucleotide primers (IDT, Coralville, IA). The ydiF gene was cloned into a modified pET15b vector (Amersham Biosciences) and expressed in E. coli BL21(DE3) as a fusion with a TEV protease-cleavable N-terminal (His) 8 tag. The E. coli methionine auxotroph strain DL41(DE3) was transformed by the plasmid, for the production of selenomethionine-labeled protein (19).
Gel filtration chromatography was carried out using a Superose 12 HR10/30 column on an Akta Purifier FPLC system (Amersham Biosciences). Purified YdiF enzyme (200 g) was applied to the column pre-equilibrated with buffer (20 mM Tris-HCl, pH 8, 0.4 M NaCl, 5% (v/v) glycerol, 5 mM dithiothreitol) and protein elution was monitored by UV absorption at ϭ 280 nm. Molecular masses were estimated by comparison with the elution profile of molecular mass standards (Sigma). Dynamic light-scattering measurements were done on a DynaPro plate reader molecular sizing instrument (Protein Solutions, Charlottesville, VA) at room temperature using a protein concentration of 8 mg/ml in 20 mM Tris-HCl buffer (pH 8), 0.4 M NaCl, 5% (v/v) glycerol, and 5 mM dithiothreitol.
Mass Spectrometry-Electron spray ionization-mass spectrometry was performed using an Agilent 1100 Series LC/MSD (Agilent Technologies, Palo Alto, CA). YdiF protein was diluted to 0.4 mg/ml in 20% (v/v) acetonitrile, 0.1% (v/v) formic acid and ionized by direct injection. The ␥-glutamyl-CoA thioester form of YdiF was prepared in a 50-l reaction consisting of 0.15 M YdiF, 50 mM Tris-HCl (pH 8.5), and 1 mM CoA thioester (acetyl-CoA, butyryl-CoA, propionyl-CoA, or crotonoyl-CoA) in the presence and absence of 20 mM sodium acetate. For identification of products, 2 l of the reaction mixture following incubation at 21°C for 2 h was injected and analysis was carried out in negative mode with the above buffer system with 10 mM ammonium acetate.
Enzyme Activity Measurements-Characterization of YdiF enzymatic activity was performed essentially according to Buckel et al. (20). A 1-ml reaction mixture containing 50 M coenzyme A derivative (Sigma), 10 mM sodium acetate (or other carboxylic acid), 10 mM oxaloacetate, 10 g of citrate synthase (Sigma), 10 mM 5,5Ј-dithiobis(nitrobenzoic acid), and 20 g of purified YdiF was incubated at room temperature for 30, 60, and 120 min and the release of free coenzyme A monitored at 412 nm and detected via formation of the nitrothiobenzoate dianion. Propionate-CoA transferase from Clostridium propionicum (21) was used as a positive control.
Crystallization-Initial crystallization conditions were determined by hanging drop vapor diffusion using screens from Hampton Research (Laguna Hills, CA). The best YdiF crystals were obtained by equilibrating 1 l of protein (7.5 mg/ml) in buffer (20 mM Tris-HCl, pH 8, 0.4 M NaCl, 5 mM dithiothreitol) mixed with 1 l of reservoir solution (22.5% (w/v) polyethylene glycol 4000, 3% (v/v) isopropyl alcohol, 0.1 M Hepes, pH 7.5) and suspended over 1 ml of reservoir solution. Crystals grew to a size of ϳ0.1 ϫ 0.1 ϫ 0.06 mm in 2 days at 21°C. For data collection, crystals were transferred for 1 min to a cryo-protectant solution containing reservoir solution supplemented with 17% (v/v) 2-methyl-2,4pentanediol, picked up in a nylon loop, and flash cooled in a N 2 cold stream (Oxford Cryosystem, Oxford, UK). Crystals of YdiF belong to the space group P2 1 with unit cell dimensions a ϭ 80.2, b ϭ 132.3, c ϭ 105.1 Å, ␤ ϭ 100.6°, and Z ϭ 8, with a V m of 2.4 Å 3 Da Ϫ1 and a solvent content of 48% (22).
Crystals of the YdiF-␥-glutamyl-CoA thioester were obtained by cocrystallization of YdiF with 10 mM acetyl-CoA, acetoacetyl-CoA, propionyl-CoA, butyryl-CoA, or crotonoyl-CoA (Sigma), and reservoir solutions containing 15.5-17.5% (w/v) polyethylene glycol 3350 and 80 mM sodium potassium tartrate. Crystals formed after 3-4 days by mixing 1 l of 7.5 mg/ml YdiF in buffer and 1.5 l of reservoir solution in a microbatch plate and layering it with paraffin oil. Crystals were cryoprotected by a brief transfer to a solution containing reservoir solution supplemented with 20% (v/v) glycerol and flash cooled in the nitrogen stream at 100 K. Crystals obtained in the presence of acetoacetyl-CoA, acetyl-CoA, and propionyl-CoA also belonged to space group P2 1 with unit cell dimensions a ϭ 81.1, b ϭ 140.2, c ϭ 112.6 Å and ␤ ϭ 108.2°, whereas crystals obtained from butyryl-CoA and crotonoyl-CoA had cell dimensions a ϭ 80.8, b ϭ 137.1, c ϭ 110.4 Å, and ␤ ϭ 105.8°.
Data Collection, Structure Solution, and Refinement-Diffraction data from a selenomethionine-labeled YdiF crystal were collected using a three wavelength MAD regime with a Quantum-4 CCD detector (Area Detector Systems Corp., San Diego, CA) at beamline X8C at the National Synchrotron Light Source, Brookhaven National Laboratory. Data processing and scaling was performed with HKL2000 (23) (TABLE ONE). Of 44 expected selenium atoms in the asymmetric unit, 39 were located using data to 2.7-Å resolution with the program SOLVE (24), and used to calculate phases with a resulting figure of merit of 0.55. Density modification with the program RESOLVE (25) improved the quality of the map (figure of merit ϭ 0.73) and allowed for automated model building of 52% of main chain atoms and fitting of 26% of the expected side chains within the asymmetric unit. The partial model obtained from RESOLVE was extended manually with the help of the program O (26) and improved by several cycles of refinement using the SCHEME 1. General reaction catalyzed by CoA transferases. R and RЈ refer to the donor and acceptor acyl groups exchanged in the reaction, respectively. YdiF Acyl-CoA Transferase DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 program REFMAC (27). Neither non-crystallographic symmetry restraints nor a -cutoff were used during refinement. The histidine tag and residues 1-3, 277-283, and 343-348 were disordered in the electron density map. The final model of apo-YdiF includes four independent monomers, each consisting of residues 4 -276, 284 -342, and 349 -529 with good stereochemistry (PRO-CHECK, Ref. 28). The model also includes 1271 water molecules and has an R-factor of 0.187 and R free of 0.221 for all data to 1.9-Å resolution (TABLE ONE).
Diffraction data for YdiF co-crystallized with various CoA thioesters were collected at beamline X29, National Synchrotron Light Source, using a Quantum-315 CCD detector (ADSC). Datasets were obtained as follows: acetoacetyl-CoA (2.4 Å), acetyl-CoA (2.0 Å), propionyl-CoA (2.1 Å), butyryl-CoA (2.15 Å), and crotonoyl-CoA (2.4 Å). The structures of YdiF-CoA complexes were determined by molecular replacement using the program MOLREP (29) with the apo-YdiF tetramer as the search model. Comparison of electron density maps for each of the datasets collected showed very similar features in the active site region, therefore, only data from acetyl-CoA and butyryl-CoA co-crystals, which showed good density for CoA, were used to build and refine models of the CoA complex. In subunit D of the YdiF-CoA complex obtained from the butyryl-CoA co-crystals the C-terminal domain is less well ordered because of few crystal lattice contacts. These models were refined using REFMAC to a final R-value of 0.184 (R free of 0.224) for the CoA thioester complex derived from acetyl-CoA, and an R-value of 0.186 (R free of 0.235) for the same complex derived from butyryl-CoA, respectively. Final refinement statistics are shown in TABLE ONE.
As CoA transferases can exhibit a broad activity profile toward different CoA donors and acceptors (20,39,40), various acyl-CoA thioesters were tested for in vitro activity with YdiF. Among the CoA derivatives tested, acetoacetyl-CoA exhibited the highest activity with acetate as an acceptor. When acetyl-CoA was used as the donor, YdiF utilized propionate, acetoacetate, butyrate, isobutyrate, and 4-hydroxybutyrate as acceptors but not isovalerate (TABLE TWO). No free CoA could be detected when the enzyme was incubated with CoA derivatives in the absence or presence of co-substrate. Overall, the activity profile of YdiF with various CoA thioesters resembles that of ACT (39). Based on the activity profile and sequence analysis, we speculate that YdiF plays a role in short-chain fatty acid metabolism in E. coli (41,42).

Monomer Structure
The asymmetric unit contains four nearly identical YdiF monomers, with any pair of them superimposing with a root mean square deviation between 0.26 and 0.41 Å for all C␣ atoms. Each YdiF monomer consists of two domains, an N-terminal domain (Val 4 -Pro 254 ) and C-terminal domain (Leu 285 -Ala 529 ), each having an open ␣/␤-protein fold. A polypeptide linker (Asp 255 -Pro 284 ) connects these two domains. The N-terminal domain is made of three layers, with the core being a central, eight-stranded parallel ␤-sheet with one anti-parallel edge strand. On one side of this sheet, near its center, are two ␣-helices flanked on either side by a 4-stranded and 3-stranded mixed ␤-sheet, respectively, together forming a second layer. The third layer, on the opposite side of the central ␤-sheet, is made of three ␣-helices and a short helical turn (Fig. 1a). The C-terminal domain has a very similar three-layered architecture with a central, 10-stranded mixed ␤-sheet with three ␣-helices and a ␤-hairpin on one side forming the domain interface, and two ␣-helices and a helical turn on the other, solvent-exposed side. The two domains associate to form a bowl-like shape with a deep cleft between them, and the active site located at the bottom of the bowl. The three-dimensional structure clearly indicates an ancestral gene duplication event. The N-and C-terminal domains can be superimposed with a r.m.s. deviation of 1.6 Å for 85 C␣ pairs. Structure-based sequence alignment of these two domains shows several long insertions in different locations (result not shown). The very low level of sequence identity retained between the two domains (ϳ6%) suggests that this gene duplication event occurred in the distant past. A similar ancient gene duplication event has been postulated for the ␣and ␤-subunits of GCT, which together form the active heterodimer (14).

Quaternary Structure
YdiF forms tetramers in solution, as determined by both gel filtration and dynamic light scattering studies. The crystal structure shows that the YdiF tetramer is formed as a dimer of dimers having pseudo 222 symmetry, with the two dimers (AB or CD) associating tightly along the pseudo 2-fold axis (Fig. 1b). The contacts between dimers are less pronounced than those involved in dimer formation, with a buried area of ϳ1470 Å 2 or ϳ4% per dimer. At the dimer-dimer interface, the N-terminal domain of each monomer (A or B, respectively) makes contacts (Ͻ4 Å) with only one monomer (C or D, respectively) of the second dimer. The dimer-dimer interface of the tetramer contains more ordered water molecules resulting in additional bridging hydrogen bonds than the monomer-monomer interface of the dimer. The associations of the monomers into a tetramer are such that the substrate binding clefts of each monomer remain solvent exposed.
Intermolecular contacts of the dimer involve both the N-and C-terminal domains of the protein and are predominantly van der Waals interactions with few hydrogen bonds. An intramolecular salt bridge between Arg 126 of the N-terminal domain and Asp 364 in the C-terminal domain at the center of the dimer interface contributes to stabilization. The surface area buried as a result of dimerization is ϳ2,600 Å 2 per monomer, corresponding to 12% of the total monomer surface area. The two independent dimers can be superimposed with a r.m.s. deviation of 0.28 Å, identical to that for individual monomers, indicating a

Complex with Coenzyme A
To define the substrate binding site and residues involved in catalysis we co-crystallized YdiF with several coenzyme A thioesters in the absence of the acceptor co-substrate, resulting in trapping of CoA in the form of its ␥-glutamyl-CoA thioester. The extent of electron density observed for CoA varied in the different subunits obtained from the various data sets. In the crystal structure of YdiF co-crystallized with butyryl-CoA, electron density corresponding to that of a covalent thioester between Glu 333 and CoA was observed in subunits A, B, and C (Fig.  2a). In these three subunits, the phosphoadenosine moiety showed stronger electron density compared with that for the pantetheine moiety. In subunit D, the electron density was weaker for both the phosphoadenosine as well as the pantetheine moieties, and density consist- ent with a covalent thioester was not observed. In YdiF co-crystallized with acetyl-CoA, electron density for the thioester linkage could be observed in the A and C subunits. Good density for the phosphoadenosine moiety and weak density for pantetheine portion of CoA, with no continuous density to Glu 333 , was observed in subunits B and D. In subunit B, the Glu 333 side chain was weakly defined, suggesting it assumes several conformations.
CoA binds in the cleft formed at the interface of the N-and C-terminal domains, with all interactions with CoA coming from the C-terminal domain (Fig. 2b). The interactions between YdiF and CoA are the same in all subunits. The CoA binding pocket is formed by residues 306 -311, an extended "flap" (389 -402) and residues 419 -423 and 440 -442. Binding of CoA results in localized structural changes for residues 300 -315 and 410 -430 and the side chains of Arg 288 and Phe 392 . Superposing the tetramers of apo-YdiF and the CoA thioester complex gives a r.m.s. deviation of 0.6 Å for all main chain atoms indicating that CoA binding causes no large structural changes.
The portion of CoA making the most abundant protein interactions is the diphosphate moiety, which is hydrogen-bonded to the side chains of Arg 288 and Ser 377 , to the main chain amide of Ile 311 and through bridging waters to the NH groups of Phe 378 and Thr 417 , the carbonyl of Cys 415 , and the side chains of Lys 442 and Thr 417 (Fig. 2b). The O-2Ј atom of ribose is hydrogen bonded to the NH group of Gly 421 . Finally, the adenine N-6 atom forms hydrogen bonds to the backbone carbonyl of Ala 379 and through a bridging water molecule to the side chain of Glu 380 and the carbonyl of Lys 441 , whereas the N-1 ring atom contacts Glu 380 and Asn 393 through water molecules. The adenine ring also makes a herringbone contact with the ring of Phe 392 . The pantetheine moiety predominantly makes van der Waals contacts within the mainly hydrophobic bottom part of the binding pocket (residues 309 -310, 376 -379, and 389 -405). A water-mediated hydrogen bond is observed between the pantetheine N-4 atom and the NH of Gly 401 , whereas a second water bridges the pantetheine O-5 atom with the carbonyl of Val 309 and NH of Ser 377 . Concomitant with CoA binding, the electron density for the side chains of Val 309 , Met 397 , and Ile 405 becomes somewhat more diffuse, consistent with mobility of the pantetheine portion of CoA.
Formation of the ␥-glutamyl-CoA thioester in solution was verified by electron spray ionization-mass spectrometry following incubation of YdiF with butyryl-CoA, revealing a single species corresponding to a mass of 60,379 Da, which is 751 Da in excess of the native molecular mass of 59,628 Da, with no mass corresponding to the apoprotein being observed. The excess mass corresponds well to the expected mass difference of 749 Da for the covalent ␥-glutamyl-CoA thioester formed between the thiol group of CoA and the carboxyl of Glu 333 , as supported by the crystallographic evidence herein. Detection of only the ␥-glutamyl-CoA thioester confirms that in the absence of co-substrate, the reaction stops at this intermediate, as previously observed by MS with GCT (10) and SCOT (43), or by enzymatic assay with SCOT (44,45).

Catalytic Site
In all YdiF-related CoA transferases, the sequence motifs 333 EXGXXG 338 and 398 GXGG(A/F) 402 are conserved, with the former sequence containing the catalytic glutamate residue (10,46,47) and the latter forming the oxyanion hole (14). In those subunits that show electron density for CoA, Glu 333 adopts one of two extended orientations. Where the density is consistent with formation of the ␥-glutamyl-CoA thioester, Glu 333 (conformation I) forms a water-mediated hydrogen bond to the amide of Gly 401 (Fig. 3a). In this conformation, Asn 306 is re-positioned so that it forms a hydrogen bond with the main chain atoms of Tyr 375 and CO of Val 307 . In subunit D of the complex obtained using acetyl-CoA, Glu 333 is not involved in a covalent interaction with CoA (conformation II) but forms a hydrogen bond with Gln 118 , and through a water molecule to the amide of Gly 401 (Fig. 3b). In the native structure, Glu 333 assumes a bent conformation (conformation III) in all four subunits, and is stabilized by side chain hydrogen bonds to Asn 306 and, through a bridging water molecule to Gln 118 and NH of Gly 401 (Fig.  3c). Binding of CoA, and the concomitant change in orientation of Glu 333 , results in breakage of its hydrogen bond with Asn 306 . Together, these results show that the catalytic Glu 333 in YdiF adopts three distinct conformations during the catalytic cycle.

Comparisons with Family I CoA Transferases
Overall Fold-The structures of the individual YdiF domains closely resemble those of SCOT (16), the ␣and ␤-subunits of the GCT heterodimer (14), and the ACT ␣-subunit (15), with a r.m.s. deviation of 1.4 -1.6 Å for the C␣ atoms in pairwise structural alignments. When full-length YdiF and SCOT monomers are superimposed, the r.m.s. deviation is greater, because of a small difference in the relative orientation of the domains connected by a flexible linker. These domains are grouped into the NagB/RpiA/CoA transferase fold in SCOP (48) sharing a common ␣/␤/␣ architecture and a central 6-stranded ␤-sheet. The ␣and ␤-subunits are classified into individual superfamilies within this fold.
CoA Binding Site-Comparing the CoA binding region of YdiF with SCOT (C-terminal domain, Protein Data Bank code 1OOY) and ␤-GCT (subunit, PDB 1POI) reveals that spatially similar elements of secondary structure interact with CoA. Structurally similar residues involved with CoA binding, in addition to the catalytic glutamate residue, Glu 333 (Glu 305 of SCOT or Glu 54 of ␤-GCT), include Arg 288 , Val 307 , Gly 308 , Gly 310 , Leu 376 , Ala 379 , Phe 392 , Gly 400 , Gly401, Ile 405 , and Lys 442 (Fig. 3d). Several of these residues are in the vicinity of the pantetheine moiety. Based on these observations, both SCOT and GCT would be expected to exhibit similar binding interactions with CoA as does YdiF. However, the structural superposition indicates that SCOT would require an inter-domain movement to effectively interact with CoA, as has been suggested earlier (11).
Co-substrate Binding Site-Little experimental data are available about specific residues of Family I CoA transferases that are involved in co-substrate binding. Comparison of the active site regions of YdiF, SCOT, and GCT suggests that the residues likely to be involved in co-substrate binding differ among these enzymes. In YdiF, these include the structurally conserved residue Gln 118 , and the non-conserved residues Gly 37 , Thr 69 , Gly 70 , His 95 , and Gln 99 . Additional residues proposed to participate in co-substrate binding in GCT (14) are part of the insertion region (76 -84) and are absent in YdiF. The shorter 341-347 loop in YdiF results in the cleft being more open and accessible to the cosubstrate, whereas in contrast, the longer loops in SCOT and ␤-GCT results in narrowing of the cleft.
Mechanism of Action-In Family I CoA transferases, the catalytic transfer of coenzyme A from the acyl-CoA thioester to the carboxylic acid co-substrate occurs by two half-reactions in a ping-pong kinetic mechanism (40,49) with the formation of a covalent thioester interme-diate between coenzyme A and the active site glutamate residue (7). The reaction mechanism has previously been investigated in detail, and determined to consist of several steps (Fig. 4). In the first step, the glutamate side chain attacks the carbonyl carbon of the thioester linkage, resulting in breakage of the CoA thioester bond and formation of a glutamyl anhydride intermediate (A). In the second step, the sulfur anion of CoA attacks the carbonyl carbon of the catalytic glutamate resulting in a covalent ␥-glutamyl-thioester intermediate (B) and concomitant release of the donor carboxylic acid. In the third step, the carboxyl oxygen of the acceptor carboxylic acid co-substrate attacks the carbonyl carbon of the glutamate side chain, liberating CoA from the glutamyl-thioester intermediate and generating a second anhydride intermediate (C). In the final step, the sulfur anion of CoA attacks the carbonyl carbon of the acceptor carboxylic acid and forms an acyl-CoA, leaving glutamate in its starting state.
From the crystal structure of the YdiF-CoA complex it is seen that three residues, Gln 118 , Asn 306 , and Glu 333 play a crucial role in the CoA transferase reaction. The principle role of Gln 118 is proposed to be in the stabilization of the catalytic glutamate residue in a conformation suitable for formation of the anhydride intermediate with the carboxylic acid. In this second conformation, the side chain of Glu 333 points toward the predicted co-substrate binding site, where it would need to form an anhydride during the catalytic cycle. The two conformations of Glu 333 differ from that found in the native structure (Fig. 3). Whereas only a single conformation of Glu 333 is found in apo-YdiF, the corresponding catalytic Glu 305 in apo-SCOT displays different conformations in different subunits, which correspond well to the three conformations in the various YdiF structures. Asn 306 is involved in stabilizing Glu 333 in its resting position when no acyl-CoA is bound. However, during the formation of the ␥-glutamylthioester intermediate Asn 306 assumes a different orientation. The movement of Glu 333 from conformation I to II as a covalent thioester results in re-positioning of Asn 306 and in the changes in hydrogen bonding interactions that we observe (Fig. 3). Because of the absence of bound CoA in SCOT, only one orientation of Asn 281 , equivalent to Asn 306 of YdiF, is observed, regardless of the conformation of the catalytic Glu 305 (PDB 1M3E, Ref. 16).
Based on these findings, the structural basis for the mechanism of action of Family I CoA transferases is proposed. Upon binding of the acyl-CoA, Glu 333 re-orients from its resting position (conformation III) to adopt an extended conformation (II) with a concomitant shift in the main chain atoms of the 332-334 loop. This would favor attack on the carbonyl carbon of the thioester leading to formation of an anhydride intermediate between Glu 333 and carboxylic acid. As previously proposed (14), the oxyanion hole in YdiF would serve to neutralize the developing negative charge in the transition state. The anhydride intermediate would be stabilized by a hydrogen bond to Gln 118 . Attack of the sulfur anion at the side chain carbonyl carbon of Glu 333 results in formation of the covalent thioester intermediate, repositioning of Glu 333 from conformation II to I, as well as movement of the 306 -312 loop and of the pantetheine moiety of CoA (Fig. 3). Binding of the co-substrate initiates the second half-reaction, and movement of Glu 333 from con-formation I to II. The remaining steps are essentially an inverse of the first half-reaction.
Biochemical evidence for the formation of an enzyme-bound covalent ␥-glutamyl-CoA thioester intermediate for Family I CoA transferases has been provided previously (7,44). Here, we complement and extend previous studies by employing x-ray crystallography to view the molecular details of the ␥-glutamyl-CoA thioester intermediate. It has been shown that the pantetheine portion of CoA destabilizes the E-CoA covalent intermediate, but stabilizes the transition state, together resulting in an acceleration of the second half-reaction in SCOT (12,13). In contrast, binding of the nucleotide portion of CoA has been shown to be strongly stabilizing in both the E-CoA intermediate and transition states, and weak in the Michaelis complex. The function of the nucleotide portion of CoA has been described as to "pull " the pantetheine moiety into the active site where it becomes highly reactive (12). Structural evidence consistent with these results is provided by the present structure where we observe that the electron density for the nucleotide portion of CoA is always stronger, and therefore better ordered, than that of the pantetheine moiety. In the YdiF-CoA complex, the polar atoms of the pantetheine moiety are surrounded mainly by a hydrophobic environment, which may account for at least part of the destabilizing effect of this group in the E-CoA intermediate.

Conclusions
In this study we have trapped the CoA thioester intermediate of YdiF, and compared the CoA binding site to those of other Family I CoA transferases. Clear similarities in the modes of CoA recognition by all these enzymes are evident, although there are structural differences in their co-substrate binding sites. It is clear from this study that the catalytic glutamate changes its conformation along the reaction pathway that differs between the unbound state, anhydride, and thioester intermediate, and helps to rationalize the previously observed multiple conformations of the catalytic glutamate in the structures of SCOT and GCT. The previously suggested mobility of the pantatheine moiety of CoA, supported by our crystallographic studies, plays an important role in catalysis and is expected to be observed in other members of Family I CoA transferases. YdiF Acyl-CoA Transferase DECEMBER