Structural Basis for the ATP-dependent Configurationof Adenylation Active Site in Bacillus subtiliso-Succinylbenzoyl-CoA Synthetase*

Received for publication, July 2, 2015, and in revised form, August 8, 2015 Published, JBC Papers in Press, August 14, 2015, DOI 10.1074/jbc.M115.676304

Yaozong Chen, Yueru Sun, Haigang Song, and Zhihong Guo1

From the Department of Chemistry and State Key Laboratory for Molecular Neuroscience, Hong Kong University of Science andTechnology, Clear Water Bay, Kowloon, Hong Kong SAR, China

Background: It is not clear how the adenylation active site is formed in adenylating enzymes.Results: ATP binding induces adenylate-forming conformation, creates a binding site for the carboxylate substrate, and alignsactive site residues for catalysis.Conclusion: ATP configures the adenylation active site.Significance: A new structural role is revealed for ATP in forming the active adenylation conformation in the catalysis ofadenylating enzymes.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essentialadenylate-forming enzyme targeted for development of novelantibiotics in the menaquinone biosynthesis. Using its crystalstructures in a ligand-free form or in complex with nucleotides,a conserved pattern is identified in the interaction between ATPand adenylating enzymes, including acyl/aryl-CoA synthetases,adenylation domains of nonribosomal peptide synthetases, andluciferases. It involves tight gripping interactions of the phos-phate-binding loop (P-loop) with the ATP triphosphate moietyand an open-closed conformational change to form a compactadenylation active site. In MenE catalysis, this ATP-enzymeinteraction creates a new binding site for the carboxylate sub-strate, allowing revelation of the determinants of substrate spec-ificities and in-line alignment of the two substrates for backsidenucleophilic substitution reaction by molecular modeling. Inaddition, the ATP-enzyme interaction is suggested to play a cru-cial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction hasalso clarified the positioning and catalytic role of a conservedlysine residue in stabilization of the transition state. These find-ings provide new insights into the adenylation half-reaction inthe domain alteration catalytic mechanism of the adenylate-forming enzymes.

o-Succinylbenzoyl-CoA synthetase, or MenE, is one of theenzymes responsible for biosynthesis of vitamin K in bacteria,cyanobacteria, algae, and plants (1, 2). It converts OSB2 to OSB-

CoA and is essential in a number of bacteria that rely on vitaminK2, or menaquinone, for electron transportation in their respi-ratory chains, including microbial pathogens such as Haemo-philus influenzae and Staphylococcus aureus (3–5). Thisenzyme is absent in mammals and thus is an attractive target fordevelopment of new antibacterial agents. In recent years, anumber of potent inhibitors of MenE have been synthesized,and some of them have been shown to exhibit high antituber-cular activities (6 – 8).

MenE is homologous to acyl/aryl-CoA synthetases (ACS)and catalyzes a similar two-step reaction. In the first adenyla-tion step, the OSB substrate reacts with Mg2�-ATP to form theadenylate intermediate, OSB-AMP, which subsequently reactswith the third substrate, coenzyme A, to form OSB-CoA in thesecond thioesterification step (Fig. 1). The same adenylationstep is found in the reactions catalyzed by the adenylationdomains of nonribosomal peptide synthetases (NRPS) and fire-fly luciferases, which together with acyl/aryl-CoA synthetasesform the ANL family of adenylating enzymes (9). Over the lastseveral decades, several members of this enzyme family, includ-ing MenE, have been kinetically analyzed to adopt an ordered BiUni Uni Bi ping-pong mechanism, which involves ATP as thefirst binding substrate and a large conformational change dur-ing the catalysis (6, 10 –15).

Early structural and related biochemical studies of adenylat-ing enzymes in the ANL family were focused on luciferases andthe adenylation domains of NRPS, which were found to behighly conserved in the three-dimensional structure consistingof a large N-terminal domain (N-domain) containing �400 –550 residues and a smaller C-terminal domain (C-domain) con-taining �130 residues (16 –18). Ten conserved motifs, namelyA1–A10, were identified to play important structural and cat-alytic roles in the enzymatic reactions, of which the A3 motif isthe most conserved among the ANL family (19). This conservedA3 motif overlaps with “motif I,” which defines a consensus

* This work was supported by Grants GRF601413 and N_HKUST621/13 fromthe Research Grants Council and Grant SBI14SC05 from the UniversityGrants Council of the Government of the Hong Kong Special Administra-tive Region. The authors declare that they have no conflicts of interest withthe contents of this article.

The atomic coordinates and structure factors (codes 5BUQ, 5BUR, and 5BUS)have been deposited in the Protein Data Bank (http://wwpdb.org/).

1 To whom correspondence should be addressed. Tel.: 852-23587352; Fax:852-23581594; E-mail: chguo@ust.hk.

2 The abbreviations used are: OSB, o-succinylbenzoyl; r.m.s.d., root meansquare deviation; ACS, acyl/aryl-CoA synthetase; NRPS, nonribosomal pep-tide synthetase; PDB, Protein Data Bank; AMPCPP, �,�-methyleneadeno-

sine 5�-triphosphate; 4CBL, 4-chlorobenzoyl:coenzyme A ligase; LC-FACS,long chain fatty acyl-CoA synthetase.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 39, pp. 23971–23983, September 25, 2015

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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sequence of (T/S)(S/G)G(T/S)(T/S/E)GX(P/Q/M/T)K (20) andis homologous to the conserved phosphate-binding loop(P-loop) involved in binding of triphosphate by ATP- and GTP-binding kinases (21). However, the active site formed at theinterface of the N- and C-domains in the early crystal structuresis only suitable for the first-step formation of the adenylateintermediate from the carboxylate and ATP substrates.

In 2003, Gulick and co-workers (22) used a nonhydrolyzableadenylate, adenosine 5�-propyl phosphate, and coenzyme A totrap the bacterial acetyl-CoA synthetase in a different confor-mation in which the C-domain is rotated around a hinge resi-due by 140° to form a new active site suitable for the second-step thioesterification. On this basis, a domain alterationmechanism was proposed for all adenylating enzymes in theANL family involving two distinct active conformations, onefor adenylate intermediate formation and the other for thioes-ter formation (9). These two conformations form two distinctactive sites with the same set of N-domain residues but a differ-ent set of residues on two opposite sides of the small C-domain,which rotates a large angle after adenylate formation to achieveconformational change and active site re-configuration.

This domain alteration mechanism is strongly supported bythe finding that mutation of residues on a different side of theC-domain specifically affects either the first or the second stepof the reaction catalyzed by a number of adenylating enzymessuch as 4-chlorobenzoyl:coenzyme A ligase (4CBL) (15), bacte-rial acetyl-CoA synthetase (23), luciferase (24), and the stand-alone adenylation domain EntE of the enterobactin synthetase(25). At present, both the adenylate-forming and thioester-forming conformations have been structurally characterizedfor several ANL family members, including DltA from Bacillussubtilis (26, 27), 4CBL (28, 29), human medium-chain acyl-CoAsynthetase 2A (ACSM2A) (30), and firefly luciferase (16, 31).

An interesting question in the domain alteration mechanismis how ATP configures the adenylation active site as the firstbinding substrate. At present, there is limited informationabout this process probably due to the difficulty to avoid autohy-drolysis in co-crystallizing ATP with the enzymes. In the fewavailable complex crystal structures (30, 32, 33), ATP or itsanalog is bound to the ANL enzymes in different modes andonly the P-loop of ACSM2A is found to form gripping interac-tions with the triphosphate of the ligand as expected. In thisstudy, we use MenE to further explore the binding and config-uration of the adenylation active site by ATP. We have solvedthe crystal structures of B. subtilis MenE (BsMenE) in a new

ligand-free form and in complex with ATP or AMP. Thesestructures have led to a general ATP binding mode and anopen-closed conformational change in ATP configuration ofthe adenylation active site.

Experimental Procedures

Chemicals—The MenE substrate OSB was prepared che-moenzymatically from chorismic acid that was isolated from anengineered Escherichia coli strain KA12 (34). It was synthesizedin one pot using recombinant EntC (35), MenC (36, 37), MenD(36, 37), and MenH (38 – 40) and purified by reverse-phaseHPLC. The isolated product was lyophilized and re-dissolved in10 mM Tris (pH 8.0) buffer for determination of its concentra-tion by the UV absorbance at 250 nm (the extinction coefficient� � 4900 M�1 cm�1). Other chemicals used in this study,including ATP, AMP, CoA, NaHCO3, isopropyl �-D-1-thioga-lactopyranoside, polyethylene glycol (PEG), (�)-2-methyl-2,4-pentadiol, buffers, and other salts, were purchased from Sigma.

Expression and Purification of BsMenE and Its Mutants—The menE gene was amplified from the genomic DNA of B.subtilis strain 168 using 5�-ATT TGC GGC CGC ATG CTGACA GAA CAG CCC AA-3� and 5�-CCG CTC GAG TCATAG CAG TTC TCC TTT ACG-3� as primers and was insertedinto pET28a between NotI and BamHI sites. The subclonedgene was sequenced by Beijing Genomics Institute (BGI, Shen-zhen, China) and found to be identical to the sequence depos-ited in GenBankTM (accession number NP_390957). The pro-tein with a C-terminal hexahistidine tag was overexpressed inE. coli strain BL21 (DE3) in Luria broth (LB) containing 25�g/ml kanamycin for 8 h at 37 °C after induction by 0.2 mM

isopropyl �-D-1-thiogalactopyranoside and purified to homo-geneity by Ni2�-chelating column chromatography and sizeexclusion chromatography (HiPrep 16/60 Sephacryl, GEHealthcare). The purified protein was concentrated, flash-fro-zen in liquid nitrogen, and stored at �20 °C in 10 mM Tris-HCl(pH 8.0) buffer containing 2 mM 2-mercaptoethanol and 10%glycerol. The protein concentration was determined byPierceTM Coomassie Plus (Bradford) assay kit (ThermoScientific).

The expression constructs of BsMenE mutants R382A,R382K, T152A, G154P, T155A/T156A, T155A, T156A, andG157P were generated by QuikChangeTM site-directedmutagenesis kit (Stratagene) using the BsMenE-expressingplasmid in pET28a as template. All mutants were sequenced byBGI to confirm that only the desired mutations had been incor-

FIGURE 1. Reaction catalyzed by OSB-CoA synthetase (MenE). A, MenE-catalyzed two-step reaction. B, ordered Bi Uni Uni Bi ping-pong catalyticmechanism.

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porated into the menE gene. The expression and purification ofthese mutants followed the same protocol described above forthe wild-type BsMenE.

Activity Assay and Steady-state Kinetics—The BsMenE activ-ity was determined by a coupling assay using E. coli MenB (6),which was obtained as an untagged recombinant protein asreported previously (41, 42). In the assay, the BsMenE productOSB-CoA was transformed by excess MenB to 1,4-dihydroxyl-2-naphthoic acid-CoA for measurement at 392 nm (molarextinction coefficient � � 4000 M�1cm�1) (43). For the singlesubstrate kinetics, a 140-�l standard assay mixture contained50 mM Tris-HCl (pH 7.5), 20 mM NaCl, 5 mM MgCl2, 80 �M

MenB, 20 mM NaHCO3 and a varied concentration of the sub-strate OSB, ATP, or CoA with the other two substrates fixed ata saturation concentration (OSB at 1.0 mM, ATP and CoA at 2.0mM). The reactions were initiated by addition of BsMenE to afinal concentration of 250 nM after a 3-min incubation. The realtime absorbance increase at 392 nm was measured for at least 1min, and the reaction rate was calculated from the slope of theinitial linear region of the reaction progress curve. All activityassays were carried out in triplicate at 24 °C. Subsequently, thedata were fit to Michaelis-Menten Equation 1,

v � Vmax�S/KM � �S� (Eq. 1)

where � is the reaction rate (in �M/min); Vmax is the maximumreaction rate (in �M/min); [S] is the substrate concentration (in�M), and KM is the Michaelis-Menten constant (in �M).

Crystallization—Crystallization conditions were screenedusing Index Screen and Crystal Screen I and II from HamptonResearch or WizardTM Classic 3 and 4 from Rigaku. Crystalli-zation was carried out with the hanging drop vapor diffusionmethod at 16 °C. A rod-like crystal (150 � 20 � 30 �m) ofligand-free BsMenE was obtained after a 10-day growth in a 1:1mixture of a solution of BsMenE at 10 mg/ml and a reservoirsolution at pH 6.15 containing 8.1% (w/v) PEG8000, 0.09 M

cacodylate/HCl, 0.144 M calcium acetate, 12.6% glycerol, 0.049M sodium phosphate monobasic, and 0.091 M dibasic potassiumphosphate. A trapezoid-like crystal (200 � 100 � 70 �m) ofBsMenE�ATP-Mg2� complex was grown within a week in a 1:1mixture of a solution of 10 mg/ml BsMenE preincubated with 3mM ATP and 5 mM MgCl2 and a reservoir solution at pH 7.7containing 7.2% (v/v) ethylene glycol, 9.9% (w/v) PEG8000, 0.09M HEPES, 3.5% (v/v) 2-methyl-2,4-pentanediol, and 0.01 M ace-tate. Single hexagonal crystals of the BsMenE-AMP complexwith the longest dimension up to 50 �m were grown in the 1:1mixture of a solution of 10 mg/ml BsMenE preincubated with 3mM AMP and a reservoir solution at pH 7.5 containing 0.10 M

HEPES, 10% (w/v) PEG6000, 5% (v/v) (�)-2-methyl-2,4-penta-diol. These crystals were soaked in cryoprotectant containing20% (v/v) glycerol in the mother liquor and then flash-frozen inliquid nitrogen before data collection.

Data Collection, Processing, and Structural Determination—X-ray diffraction data were collected for the obtained BsMenEsingle crystals at the beamline BL17U of the Shanghai Synchro-tron Radiation Facility with an ADSC Quantum 315R charge-coupled device detector. Diffraction images were indexed, inte-grated, and scaled using HKL2000 and iMOSFLM in the CCP4

suite (44). TRUNCATE of the CCP4 package was used to con-vert the intensities to structure factors. The ligand-freeBsMenE structure was solved by molecular replacement withPhaser (45) in the CCP4 suite using the backbone of the N-do-main (residue 1–394) in chain A of S. aureus MenE (PDB code3IPL) as the search model. The initial electron density mapshowed that two BsMenE polypeptide chains were correctlylocated in the asymmetric unit in the space group P1211 forthe apo-form. After refinement by PHENIX (47), theC-domain(383– 486) of apo-MenE was extended via manualmodel building using Coot (48) followed by refinement withREFMAC (49) and PHENIX. When the refinement was close tocompletion, TLS anisotropic refinement (50) was performedfor both the N- and C-domains. For the binary complex withAMP and ternary complex with ATP and Mg2�, the diffractiondatasets were processed similarly, and chain A of the full-lengthapo-BsMenE was used as the search model for structural deter-mination by molecular replacement. Both structures weredetermined to have a space group of P43 and refined using thesame strategy as for the apo-protein. Restraints of ATP andAMP were generated and refined using eLBOW (51). The over-all quality of the three structural models was assessed by PDBvalidation, Procheck (52), and MolProbity (53). Statistics for thedata collection and structural refinement are summarized inTable 1.

Structural Analysis and Sequence Alignment—BsMenE ho-mologs were identified using a PSI-BLAST search and alignedwith ClustalW 2.0 (54). Multiple sequence alignments were dis-played with ESPript 3.0 (55). PyMOL version 1.3 was used in thestructural analysis and generation of all graphics (56). PISA wasused to analyze the protein interfaces (57), and CASTp wasused to identify the cavities in the protein structures and tocalculate their volume (58).

Docking Simulation—All docking simulations were run withAutoDock Vina 1.1.2 using the ATP-BsMenE complex assearching macromolecule (59). The grid maps with 1.000 Åspacing were generated by AutoDock Tools (ADT) with thecenter set at the coordinates 54.113, 157.401, and 8.502 in the x,y, and z directions, respectively, and the grid box was set at a sizeof 14, 6, and 6 for the three dimensions. Hydrogen bonds andvan der Waals interactions were modeled with a Lennard-Jonesparameter of 12–10 and 12– 6, respectively. The electrostaticgrid maps were calculated using the distance-dependent dielec-tric permittivity of Mehler and Solmajer (60). The docking sim-ulation was run independently 10 times, and the resulting con-formations within a root mean square deviation (r.m.s.d.) of 2.5Å were clustered and analyzed.

Results

Overall Structure—The crystal structures of ligand-free,AMP-bound, and ATP-bound BsMenE were determined andrefined to a resolution of 1.98, 2.60, and 2.82 Å, respectively(Table 1). The structural models have good overall stereochem-istry with most residues in the most favorable or allowedregions of the Ramachandran diagram. Ser-293 is the onlyRamachandran outlier in every subunit of all three structures,which also takes the same unfavorable conformation in otherBsMenE structures determined previously (61). Curiously, the

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corresponding residue of BsMenE Ser-293 is also a Ramachan-dran outlier in the resolved crystal structures of other adeny-late-forming enzymes (22). In the three new BsMenE struc-tures, two substantially different polypeptide chains withoutthe histidine tag and the first two residues at the N terminus arepresent in the asymmetric units. These two subunits form aninterface with extensive hydrogen bonding and ionic interac-tions that are judged to be biologically relevant by PISA (Fig.2A) (57). This dimeric assembly in the crystal structures is con-sistent with the homodimeric quaternary structure determinedin solution by size exclusion column chromatography.

Both BsMenE subunits in the ligand-free structure (apo-BsMenE) contain a large N-domain (residues 3–380) and asmall C-domain (residues 387– 486), whereas only one full-length subunit (chain A) is found in the nucleotide-boundstructures. The other subunit contains only the large N-domainwith many disordered loops in both ATP-BsMenE and AMP-BsMenE structures. A significantly higher number of residuesare disordered in the linker region between the two domainsand loops in the small C-domain in the ligand-free subunitsthan in the full-length nucleotide-bound subunits of the othertwo structures. Besides the nucleotide ligand, an ethylene glycolmolecule from the crystallization buffer is found in a cavity nextto ATP in the ATP-BsMenE structure. Interestingly, the C-do-main takes a very different orientation relative to the N-domainto form a “closed” conformation in chain A and an “open” con-formation in chain B in the ligand-free structure. By compari-son, both nucleotide-bound BsMenE structures take a closedconformation similar to chain A of the apo-structure (Fig. 2B).

Open bsMenE Conformations—The open form of the ligand-free BsMenE (chain B of the apo-structure structure, Fig. 2) is anew addition to the several open conformations found in bothsubunits of another apo-BsMenE structure in a different spacegroup and the sulfate-bound BsMenE structure, which are dif-

ferent in the distance between the small C-domain and the largeN-domain (61). Although it is not possible to compare this newopen apo-BsMenE structure with the previously determinedstructures due to unavailability of the latter in the Protein DataBank, the multiplicity of the open conformations clearly showsthe intrinsic property of the enzyme to be present in thesestructural forms in the absence of a ligand. Noticeably, a similaropen conformation is also present for the apo-form of anotherMenE orthologue from S. aureus subsp. aureus Mu 50 (PDBcode 3IPL), demonstrating that the open conformation is notunique to BsMenE but may be a general structural form for allMenE orthologues.

Actually, open conformations similar to those of BsMenE arealso present for other adenylate-forming enzymes (16, 34). Acommon feature of these conformations is that the small C-do-main rotates away from the N-domain at a large angle in com-parison with the closed adenylate-forming conformation.However, the rotation angle varies in a wide range and is differ-ent for each enzyme. The abundance of these open conforma-tional states clearly indicates the high conformational dynamicsof the small C-domain and strongly suggests that it is an intrin-sic property of the adenylate-forming enzymes to populate var-ious open conformations with a variable distance between itstwo domains when substrates or other ligands are absent. Intheir resting state, these enzymes may also populate closed con-formations as suggested by the closed conformations observedin the absence of a ligand for MenE (chain A of apo-BsMenE,Fig. 2B) and other adenylate-forming enzymes such as DhbE(18), 4CBL (29), and DltA (62). These resting conformationsform the structural basis for a potential substrate recognitionmechanism in which the active closed adenylate-forming con-formation is either induced or stabilized from the resting openand closed conformations by binding of the carboxylate or ATPin the first-step adenylation reaction.

TABLE 1Data collection and refinement statisticsValues for the highest resolution shell are given in parentheses.

apo-BsMenE ATP-BsMenE AMP-BsMenE

PDB codes 5BUQ 5BUR 5BUSData collection

Space Group P1211 P43 P43Unit cell dimensions

a, b, c (Å) 82.8, 76.9, 93.9 121.8, 121.8, 97.8 121.8, 121.8, 98.0�, �, (°) 90.0, 114.9, 90.0 90, 90, 90 90, 90, 90

Redundancy (%) 2.3 6.2 3.1Completeness (%) 98.83 (99.15) 99.74 (100.00) 99.46 (99.34)Reflections (unique) 141,919 (73,620) 606,644 (49,305) 2,042,714 (43,794)I/I 22.1 (9.0) 30.0 (3.7) 6.6 (4.9)Rmerge 0.048 (0.204) 0.091 (0.692) 0.136 (0.864)

RefinementResolution range (Å) 33.27–1.98 36.39–2.82 38.52–2.60No. of non-hydrogen Atoms 8104 6269 6500

Protein 7064 6146 6434Water 1035 44 62Ligands/ions 5 79 46

Average B factor (Å2) 21.00 70.90 72.70Protein 19.00 70.80 72.90Water 34.40 63.50 58.20Ligand 49.00 84.50 109.20

Rwork/Rfree 0.1605/0.1997 0.2160/0.2658 0.1904/0.2335r.m.s.d. for ideal values in

Bond length (Å) 0.008 0.011 0.009Bond angle (degree) 1.03 1.35 1.21

Ramachandran statisticsa 97.00/2.78/0.22% 93.00/6.64/0.36% 95.00/4.53/0.47%a Ramachandran statistics indicate the fraction of residues in the most favored, allowed, and disallowed regions of the Ramachandran diagram.

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Closed BsMenE Structures—All three closed BsMenE sub-units in the solved crystal structures are closely similar to theadenylate-forming conformations identified for a number ofACS, adenylation domains of NRPS, and firefly luciferases withtheir interdomain cavities corresponding to the identifiedactive sites of the adenylation step. However, they exhibit asignificant difference in the exact orientation of the C-domain(Fig. 2B), which rotates from the large N-domain in a slightlydifferent angle around the linker region between the twodomains. This difference is more pronounced between theligand-free BsMenE and the nucleotide-bound structures witha calculated interdomain cavity of 6880 Å3 for the former and6521 Å3 for the AMP-bound complex using CASTp (58),although it is much smaller between the two nucleotide-boundstructures with the smallest interdomain cavity formed in theATP-BsMenE structure. Notably, there is another closedBsMenE subunit in the previously determined crystal struc-ture in complex with the OSB-AMP intermediate (61), whichis not available for comparison. From the fact that the smallC-domain takes two very different orientations in this OSB-AMP-bound BsMenE structure as in the ligand-free BsMenEstructure, it is likely that the interdomain cavity in the OSB-AMP-bound closed conformation is similar to that in theligand-free or AMP-bound BsMenE structure and larger thanthat in the ATP-BsMenE structure.

The most compact ATP-BsMenE structure contains aligand-binding tunnel with both ends extending to the proteinsurface (Fig. 2C), which consists of two segments crossed at analmost perpendicular angle that binds the nucleoside and

triphosphate moieties of the ATP ligand, respectively. In com-parison, the binding tunnel is leaky in the AMP-BsMenE struc-ture with an additional opening to the bulk solvent, while it ishardly formed in the ligand-free closed conformation due to thedifferent C-domain orientation. The compact ATP-bindingsite is a strong indicator that the adenylation active site is prop-erly formed only in the ATP-BsMenE structure, demonstratingthe crucial role of the substrate in configuring the active site. Incontrast, the closed apo-BsMenE and AMP-BsMenE structuresare merely precursor conformations of this active conforma-tion. In the ATP-bound structure, the buffer molecule ethyleneglycol occupies a new pocket formed after ATP binding, whichshould be the binding site of the second substrate, OSB.

Nucleotide-binding Site—The nucleotide-binding site ofBsMenE is composed of many conserved amino acid residuesmostly from the large N-domain, including the highly con-served nine-residue P-loop (152TSGTTGKPK160) in the A3motif (amino acids 149 –160) (Fig. 3). As shown in Fig. 4A, theadenine ring of the ligand in the ATP-BsMenE structure makesdirect hydrophobic contact with the side chain benzene ring ofthe conserved Tyr-286 in the A5 motif oriented at an almostperpendicular angle on one side and the backbone of the tri-peptide Gly-263–Gly-264 –Gly-265 on the opposite side,although its 6-amino group forms a hydrogen bond with thebackbone carbonyl group of Ser-285. The ATP ribose takes a3�-endo pucker envelope and is stabilized by bifurcated hydro-gen bonds between the side chain carboxylate of the invariantAsp-367 in the A7 motif (amino acids 365–367) and its 2�- and3�-hydroxyl groups. The �-phosphate of ATP is bound by

FIGURE 2. Crystal structure of BsMenE. A, dimeric assembly of ligand-free BsMenE in its asymmetric unit. Chain A (green) is in a closed conformation, and chainB (brown) is in an open conformation. B, superposition of different BsMenE conformations. The open (brown) and closed (green) forms of ligand-free BsMenE arealigned with AMP-BsMenE (cyan) and ATP-BsMenE (gray) according to their N-domains (in C� lines) to demonstrate the different orientation of the C-domains(schematic). ATP of the ATP-BsMenE structure is presented as sticks to indicate the position of the interdomain active site. C, ATP bound to a tunnel with twoends open to the bulk solvent in the ATP-BsMenE structure. The complex is represented in surface and colored according to domains and conserved motifs aslabeled. The arrow denotes a probable exit route for the pyrophosphate product after the adenylation reaction. ATP is shown as sticks with the coordinatedMg2� ion denoted as a green sphere.

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hydrogen bonds with side chains of Thr-289 and Ser-153 at theN terminus of the P-loop, and likely also Lys-471 of the A10motif (amino acids 465– 474) from the C-domain with its�-NH3

� at a distance of 3.6 and 3.8 Å to one of the acidic oxygenatoms and the bridging oxygen between �- and �-phosphates,respectively. Interestingly, as shown in Fig. 4B, the �,-pyro-phosphate of the ATP ligand is fixed to the position by a largenumber of polar interactions, including hydrogen-bondinginteractions with five consecutive residues (152TSGTT156) ofthe P-loop, a salt bridge to the -phosphate with the invariantLys-160 at the P-loop C terminus, direct chelating interactionsof �- and -phosphates with the divalent Mg2� ion anchored bythe side chain carboxylate of the strictly conserved Glu-290through mediation of two water molecules, and bidendatehydrogen bonds to the �-phosphate with the positive side chainof the invariant Arg-382 from the linker A8 motif. These tightwrapping interactions for the triphosphate moiety of ATP arereminiscent of the nucleotide-binding interaction of the P-loopin ATP- and GTP-dependent kinases and are likely necessaryfor precisely positioning the �-phosphate group for in-linenucleophilic reaction with the carboxylate substrate.

AMP takes an orientation similar to ATP when bound to thenucleotide-binding site, sharing all the interactions for the ade-nine, the ribose, and the 5�-phosphate moieties but losing allthe strong interactions for the �,-pyrophosphates of the latter(Fig. 4C). Consequently, the P-loop is significantly displacedfrom its position in the ATP-BsMenE structure, and the AMPphosphate group is dislocated by 0.5 Å in the AMP-BsMenEstructure. The movement of the AMP phosphate allows it toform a 2.6 Å hydrogen bond with the imidazole ring of His-196that takes different rotameric conformations in other closedstructures. This histidine residue is equivalent to the A4 histi-dine residue conserved among long chain ACS, aryl-CoA syn-thetases, and luciferases, which corresponds to a phenylalanineresidue in the adenylation domains of NRPS and a tryptophan

residue in shorter chain ACS, and has been proposed to beinvolved in both the adenylation and thioesterification reac-tions. In the OSB-AMP-bound BsMenE structure, this histidineresidue takes another slightly different rotamer conformationby forming a hydrogen bond with the oxygen atom of the car-bonyl group linked to the phosphate group (61), suggesting thatit may be involved in binding of the carboxylate substrate, OSB,and stabilizing the transition state of the adenylation reaction.

Besides the different P-loop orientation and the associateddifferent interactions, AMP-BsMenE is different from theATP-bound structure in the absence of the ligand-mediateddomain interaction involving Arg-382, which is apparently cor-related to its incomplete closure of the interdomain active sitecavity. In the ATP-BsMenE structure, the Arg-382 side chainstrongly interacts with the ATP �-phosphate group via thebidentate hydrogen bonds with strong attractive electrostaticinteraction and thus likely provides the driving force for thecomplete active site closure. This charged residue likely exertsthis structural effect by affecting the backbone torsional anglesof the downstream hinge residue Ser-384 to move the C-do-main closer to the N-domain. Consistent with this proposedrole, this arginine residue is strictly conserved among all ANLfamily members (Fig. 3).

Comparison of ATP-bound Adenylate-forming Enzymes—Several crystal structures with bound ATP or its nonhydrolyz-able analogues have been determined for human medium chainacyl-CoA synthase (ACSM2A) (30), D-alanyl carrier proteinligase DltA from Bacillus cereus (32), and long chain fatty acyl-CoA synthetase (LC-FACS) from Thermus thermophilus (33).The LC-FACS structure is unique in taking a thioester-formingconformation with the ligand bound to a site far from theP-loop, whereas all other structures are present in the adeny-late-forming conformation with the ligand bound close to theP-loop. Among these structures, the ATP-bound BsMenE ismost similar to ACSM2A bound to ATP or AMPCPP with a

FIGURE 3. Alignment of sequences associated with substrate binding, structural changes, and transition state stabilization. Representative memberswith their PDB identification numbers in the ANL family and representative MenE orthologues are aligned separately with ClustalW 2.0, and only the relevantmotifs are presented using ESPript 3.0. The BsMenE_5BUQ in the upper alignment is identical to the MenE orthologue from B. subtilis in the lower alignment.

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similar C-domain orientation relative to the N-domain, despitea limited 19.6% sequence identity between the two proteins. Inthe ATP-ACSM2A structure (PDB code 3C5E) (30), most ofthe ligand-enzyme interactions are very similar to those in theATP-BsMenE structure and involve the same number and thesame type of interactions to the same atoms of the ligand (Fig.5). The only obvious difference is that the invariant A10 Lys-577 forms a hydrogen bond with the �-phosphate in ACSM2A

rather than a hydrogen bond to the �-phosphate of ATP by thecorresponding A10 residue in BsMenE.

Both the ATP-bound BsMenE and ACSM2A are significantlydifferent from the ATP-DltA structure in the C-domain orien-tation by as much as �48°, although they are all in the adeny-late-forming conformation (32). Their nucleotide-binding sitesalso exhibit significant differences, although they share similarligand-enzyme interactions for the adenine and ribose moieties

FIGURE 4. Stereo diagram of the BsMenE nucleotide-binding site. A, ATP in its binding site. ATP is represented as sticks (carbon atoms in green) and itsmFo � DFc omit map is contoured at 2.5 in blue mesh, and the binding site residues are represented as sticks (carbon atoms in gray). B, binding interactions forthe triphosphate moiety of ATP. This is magnified from a portion of the ATP-binding site in A and viewed in stereo from a slightly different perspective. C,comparison of AMP and ATP binding to BsMenE. The AMP-BsMenE (cyan) and ATP-BsMenE (gray) complexes are superimposed to reveal different P-looporientation, different side chain orientation of the A8 Arg-382 and A10 Lys-471, and different protein-ligand interactions at the �-phosphate. The protein-ligand interactions for the adenosine moiety of the ligands are similar in the complexes as shown in A but omitted in C for simplicity. In all three panels, theyellow broken lines denote hydrogen bonds with a distance �3.6 Å and the green spheres denote Mg2�; red dots denote bound water molecules.

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and for the invariant A8 arginine (Arg-397 in DltA) and theMg2�-chelating aspartate (Asp-298 in DltA). Specifically, thereare few hydrogen bonds between the triphosphate and theP-loop in the ATP-DltA structure, in contrast to the tight wrap-ping of the triphosphate by the P-loop in BsMenE andACSM2A. Actually, the P-loop is located at a distance from thetriphosphate in DltA and a large part of it is disordered withoutcontact with ATP. The reason for these different ATP interac-tions in DltA is unclear, and adverse crystal packing interac-tions, as implicated previously (32), are one of the possibleexplanations.

Mutation of Residues in the P-loop—The P-loop is highlyconserved among all members of the ANL family with a con-sensus sequence of (T/S)(S/G)G(T/S)(T/S/E)GX(P/Q/M/T)K(residues Thr-152 to Lys-160 in BsMenE) (20). Previous func-tional characterization of the conserved residues mainlyfocused on the penultimate proline residue and the strictly con-served glycine and lysine residues. It was found that introduc-tion of hydrophobic isoleucine or negatively charged aspartateto the positions of glycine residues causes more than a 4 ordersof magnitude decrease in catalytic efficiency in either 4CBLfrom Pseudomonas sp. strain CBS3 (20) or adenylation domainsin NRPS (19). In contrast, changes at the invariant lysine tomethionine or other polar residues are tolerated with a moder-ate decrease in catalytic activity (20, 63, 64), whereas mutationsat the proline residue cause enzyme-dependent effects thatrange from no significant activity change for a proline-to-valinechange in an NRPS adenylation domain (65) to complete inac-tivation for a proline-to-alanine mutation in 4CBL (20). Fromthe ATP-enzyme interactions in BsMenE and ACSM2A (30), itis easy to understand that the two glycine residues in the P-loophave to be conserved to avoid steric repulsion with the boundATP, whereas the proline and lysine residues are important informing the �-turn in the P-loop hairpin to tightly wrap aroundthe triphosphate of the ligand, for which the former directlyfacilitates the directional change of the polypeptide chain, andthe latter stabilizes the turn by forming hydrogen-bondinginteractions with the ligand as well as other P-loop residues. Tobetter understand the catalytic contribution of the intenseenzyme-ATP interaction (Fig. 4, A and B), the conserved ser-ine/threonine residues in the P-loop interacting with the �,-pyrophosphate of the substrate were mutated into alanine indi-vidually or in combination. For comparison, the invariant A8

Arg-382 hydrogen-bonded to the �-phosphate was mutated toalanine or lysine. In addition, proline was also introduced intothe positions of the conserved glycine residues. The resultingmutants, namely T152A, T155A, T156A, T155A/T156A,R382A, R382K, G154P, and G157P, were successfully obtainedin high purity with the same stability and circular dichroismspectra as the wild-type enzyme (data not shown), indicatingthe absence of major structural change caused by themutations.

The single-substrate kinetic constants kcat, KM, and kcat/KMof the mutants and the wild-type enzyme are listed in Table 2. Itis interesting to note that the kinetic constants are moderatelyaffected for the proline mutants of the conserved glycine resi-dues, leading to a 20 –95-fold decrease for ATP and less than a15-fold decrease for other substrates in kcat/KM in comparisonwith the wild-type enzyme. These moderate changes indicatethat the introduced proline causes a much smaller steric repul-sion in comparison with isoleucine in 4CBL (20), and it has onlya mild effect on the P-loop backbone orientation, which likelydepends more on the multiple hydrogen bonds between ATPand the P-loop residues. In contrast, the threonine-to-alaninemutations cause a much larger decrease in catalytic activity.The resulting mutants exhibit a general kcat decrease of about 2orders of magnitude for all substrates and a significant sub-strate-dependent KM increase, which is 100–200-fold for ATP,2–7-fold for OSB, and 1–6.8-fold for CoA-SH. Overall, thedecrease in the catalytic efficiency (kcat/KM) is 4800–24,000-foldfor ATP, 180 –1700-fold for OSB, and 62–190-fold for CoA-SHin single mutants and significantly higher for the double mutantT155A/T156A with a 81,000-fold decrease for ATP and a 1700-fold decrease for the other two substrates. In comparison, theR382A and R382K mutations cause similar kcat and kcat/KMdecreases for both ATP and OSB with an extent comparablewith that of the T156A mutant (Table 2). However, these twomutants cause a higher KM increase for the coenzyme A sub-strate than T156A, suggesting that Arg-382 plays a critical rolein both the adenylation and the thioesterification step. In con-trast, all the P-loop mutants mainly affect the first-stepreaction.

It is interesting to note that kcat is significantly decreased byabout 2 orders of magnitude when one or two hydrogen bondsare removed by the mutations from the numerous bindinginteractions for the �,-pyrophosphate of the ATP substrate

FIGURE 5. Structural comparison of BsMenE and ACSM2A in ATP binding. The binding site residues from BsMenE (in gray sticks) are labeled, whereas theircounterparts in ACSM2A (in yellow sticks) are presented without numbering. Hydrogen bonding interactions involving the catalytic A10 lysine (Lys-471 inBsMenE) and the A8 arginine (Arg-382 in BsMenE) are denoted by yellow broken lines.

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(Fig. 4B), demonstrating that the conserved residues not onlyare involved in ATP binding but also affect subsequent adeny-lation reaction in the catalytic process. These residues may con-tribute to the chemical reaction by keeping the ATP substratein optimal position and orientation for the reaction and by sta-bilizing the transition state through their interactions with theleaving �-phosphate group. In addition, these residues togetherwith other active site residues and the metal cofactor may alsobe involved in activating the scissile phosphorus– oxygen bondby straining it via the observed tight binding of the ATP sub-strate (Fig. 4A).

Modeling of the OSB Substrate into Its Binding Site—TheOSB substrate was successfully modeled by AutoDock Vinainto the binding site adjacent to the bound ATP in its complexwith BsMenE after removing the buffer molecule, ethylene gly-col. As shown in Fig. 6, the final model was chosen from fivestructures resulting from the docking simulations on the basisof its higher similarity to the OSB-AMP-bound BsMenE struc-ture (61) in the interactions between the OSB moiety and theenzyme. As in the adenylate-bound BsMenE structure, OSB isbound between a loop containing the tripeptide 261LLG263 withthe hydrophobic leucine side chains interacting with the ben-zene ring of the ligand and the loop containing the A4 motif(196HISG199). All these binding site residues are specificallyconserved among the MenE family members but not amongdifferent adenylating enzymes (Fig. 3). Other residues lining thesubstrate-binding site include Ser-293, Gln-294, Ser-237, Gly-287, Lys-471, and the bound ATP.

Despite their similarities, the docked structure and theadenylate-bound structure exhibit marked differences. Nota-bly, OSB in the docked structure takes a conformation with itsbenzene ring twisted at an angle to both the 2-carboxylate andthe carbonyl function of the succinyl group, whereas the OSBmoiety in the adenylate-bound structure takes a different con-

formation with its benzene ring perpendicular to the 2-carbox-ylate and coplanar with the carbonyl function of the succinylgroup. In addition, the conformation of the ethylene moiety ofthe ligand is gauche in the former and anti in the latter. Otherdifferences include the absence of hydrogen bonding of the His-196 side chain imidazole and the Ser-293 side chain hydroxyl tothe ligand in the modeled structure, which is present in theadenylate-bound structure due to rotameric flipping of the sidechain functional groups and is believed to occur in the bindingof OSB by the ATP-bound BsMenE.

In the modeled complex, one oxygen atom of the succinylcarboxylate is close to the �-phosphate of ATP with a shortoxygen-to-phosphorus distance of 3.5 Å and is opposite thepyrophosphate group of ATP. This positioning of OSB allowsbackside nucleophilic attack on the ATP �-phosphate by itscarboxylate from the other side of the pyrophosphate leavinggroup in an almost linear manner, which is consistent with thein-line reaction mechanism for the adenylation half-reactionproposed for enzymes in the ANL family. Interestingly, the �-amino group of the invariant A10 Lys-471, which has beenknown to interact with the �-phosphate of ATP (Fig. 3B), isfound to be within a hydrogen-bonding distance from the otheroxygen atom on the attacking carboxylate group in OSB. Fullyconsistent with its proposed catalytic role, the simultaneousinteraction of this invariant residue with both reacting func-tional groups gives it the best position to stabilize the transitionstate.

Discussion

Adenylating enzymes in the ANL family catalyze diversechemical transformations in cellular metabolism with two dis-tinct active sites configured by domain alteration, which is con-sistent with an ordered Bi Uni Uni Bi ping-pong kinetic mech-anism reported for various enzymes in the ACS subfamily such

TABLE 2The single substrate kinetic constants of wild-type and mutant BsMenE

Protein Substrate kcat kcat(WT)

/kcat KM KM/KM(WT) kcat/KM kcat(WT) KM/kcat KM(WT)

min�1 �M min�1�M�1

WT OSB (7.2 � 0.3) � 102 1 44 � 19 1 (1.6 � 0.6) � 107 1ATP (6.3 � 0.2) � 102 1 24 � 4 1 (2.7 � 0.4) � 107 1CoA (7.3 � 0.8) � 102 1 (2.4 � 0.8) � 102 1 (3.0 � 0.6) � 106 1

T152A OSB 8.2 � 0.7 88 (8.8 � 1.1) � 102 20 (9.4 � 0.6) � 103 1.7 � 103

ATP 8.1 � 1.2 78 (3.5 � 1.2) � 103 146 (2.3 � 0.4) � 103 1.2 � 104

CoA 12.4 � 1.5 59 (2.6 � 0.7) � 102 1.1 (4.8 � 0.8) � 104 62G154P OSB (1.3 � 0.1) � 102 5.5 (1.2 � 0.2) � 102 2.7 (1.1 � 0.1) � 106 15

ATP 99 � 14 6.3 (3.4 � 1.0) � 102 14 (2.9 � 0.6) � 105 92CoA (1.3 � 0.1) � 102 5.6 (2. 7 � 1.0) � 102 1.1 (4.8 � 1.7) � 105 6.2

T155A OSB 7.5 � 0.9 96 87 � 40 2.0 (8.6 � 2.5) � 104 1.9 � 102

ATP 4.6 � 2.5 137 (4.2 � 3.9) � 103 175 (1.1 � 0.6) � 103 2.4 � 104

CoA 6.2 � 0.6 118 (4.1 � 0.8) � 102 1.7 (1.5 � 0.2) � 104 2.0 � 102

T156A OSB 12.8 � 0.3 56 (1.4 � 0.2) � 102 3.2 (9.2 � 1.3) � 104 1.8 � 102

ATP 13.1 � 0.6 48 (2.3 � 0.2) � 103 96 (5.6 � 0.3) � 103 4.8 � 103

CoA 8.7 � 0.1 84 (2.1 � 0.3) � 102 0.9 (4.1 � 0.7) � 104 70T155A/T156A OSB 2.7 � 0.2 267 (2.8 � 0.5) � 102 6.4 (9.6 � 0.8) � 103 1.7 � 103

ATP 1.1 � 2.0 573 (3.3 � 1.1) � 103 137 (3.3 � 1.4) � 102 8.1 � 104

CoA 3.0 � 0.6 243 (1.7 � 0.9) � 103 7.1 (1.8 � 1.4) � 103 1.7 � 103

G157P OSB (2.8 � 0.2) � 102 2.6 80 � 11 1.8 (3.5 � 0.4) � 106 4.6ATP (2.4 � 0.3) � 102 27 (1.9 � 0.8) � 102 7.9 (1.3 � 0.3) � 106 20CoA (2.6 � 0.2) � 102 2.8 (2.5 � 1.2) � 102 1.0 (1.0 � 0.4) � 106 3.0

R382A OSB 9.0 � 0.2 80 57 � 10 1.3 (1.6 � 0.2) � 105 1.0 � 102

ATP 13.1 � 2.6 48 (5.1 � 1.8) � 102 21 (2.6 � 1.4) � 104 1.0 � 103

CoA 17 � 1 43 (2.7 � 0.8) � 103 11 (6.3 � 1.2) � 103 4.7 � 102

R382K OSB 13.7 � 0.8 53 66 � 19 1.5 (2.1 � 0.4) � 105 79ATP 9.8 � 0.5 64 (2.8 � 0.2) � 102 12 (3.5 � 0.4) � 104 7.7 � 102

CoA 9.7 � 4.8 75 (3.0 � 2.3) � 103 12 (3.2 � 0.9) � 103 9.3 � 102

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as MenE (6), 4CBL (15), and ACS with various chain lengthspecificities (11–14). Binding of ATP is the critical first step inthe complex catalytic mechanism of these enzymes but is notfully understood. Reported structures of three adenylatingenzymes complexed with ATP or its analogues have presentedthree different pictures for the important ATP-enzyme inter-action. In this work, we have successfully determined the struc-ture of ATP-bound BsMenE with its P-loop tightly wrappingaround the triphosphate as expected for the same structuralmotifs in ATP- or GTP-dependent kinases (21). This enzyme isfound to be highly similar to human ACSM2A in binding ATPwith all but one of the numerous ligand-enzyme interactionsconserved between the evolutionarily distal proteins. Remark-ably, all the amino acid residues involved in the ATP-enzymeinteraction are highly or even strictly conserved, suggesting anidentical ATP-binding mode and the same functional andstructural effects for ATP binding in all ANL family members.Thus, together with the previous findings for the interaction ofATP with human ACSM2A (30), this ATP-BsMenE complexstructure provides a highly conserved structural model for ATPinteraction with ANL enzymes to form the active adenylate-forming conformation. Currently, it is not clear why the nucle-otide substrate is bound to a site far from the P-loop in LC-FACS (33).

An important component of this conserved ATP-enzymeinteraction is the ligand-dependent open-closed conforma-tional change that emerges from current and numerous previ-ous studies. For BsMenE, co-existence of both open and closedconformations in the ligand-free enzyme (Fig. 2), together withthe open structures previously determined (61), is a clear indi-cation that the enzyme populates both the open and closedconformations in its resting state. Considering the tight grip-ping interactions for the ATP triphosphate that makes a 90°turn in the binding tunnel, the compact ATP-BsMenE complexis much more likely to form from an open conformation ratherthan a closed conformation similar to the active adenylate-forming conformation. On the basis of the fact that open con-

formations similar to those of BsMenE have been found in theabsence of a ligand for other adenylating enzymes in the ANLfamily (16, 34), such an open-closed conformational change isproposed to generally occur as the first step to form the adeny-late-forming active site in catalysis of all ANL enzymes. Actu-ally, a similar open-closed conformational change has beenproposed for the formation of the adenylation active site in thecatalysis of DltA, although no open conformations have beenfound for the ligand-free enzyme (26).

The slight C-domain orientation difference between AMP-and ATP-bound complexes (Fig. 2B) indicates that there existstructural elements safeguarding the formation of the compactadenylation active site in the open-closed conformationalchange. Previously, the strictly conserved A10 lysine residuewas proposed to play such a role on the basis of its hydrogenbonding to the ATP �-phosphate in ACSM2A (30). However,the A10 Lys-471 in BsMenE makes the same hydrogen-bondingcontact with the �-phosphate of the ligands as in other adeny-lating enzymes (17, 18) and is unable to account for the slightstructural difference between the two nucleotide-bound com-plexes. In contrast, the invariant A8 Arg-382 forms bidentatehydrogen bonding with the ATP �-phosphate in the ATP-BsMenE complex but makes no contact with the ligand in theAMP-BsMenE complex. This is the only residue that makesdifferential interactions in the two complexes, which mayimpact the C-domain orientation by affecting the torsionalangles of the downstream hinge residue (Ser-384 in BsMenE) inthe linker region between the N- and C-domains. This invariantA8 residue is therefore believed to be crucial for proper forma-tion of the adenylation active site, which is consistent with itspronounced effect on the enzyme activity (Table 2). Other res-idues at the domain interface are involved in the same interac-tions in the two nucleotide-bound complexes.

Another important part of the ATP configuration of theadenylation active site is to create a new binding site for thecarboxylate substrate. This new binding pocket is successfullydefined in BsMenE by molecular docking with the OSB sub-

FIGURE 6. ATP and the docked OSB in their binding sites in BsMenE. ATP and OSB are represented in green and yellow sticks for their carbon atoms,respectively. Residues contacting OSB are presented in gray sticks for their carbon atoms, and those interacting with ATP are omitted. The yellow and bluebroken lines denote hydrogen bonds and the shortest distance (3.5 Å) between the OSB succinyl carboxylate and �-phosphate of ATP, respectively.

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strate and comparison with the OSB-AMP-bound BsMenEstructure (Fig. 6), which is composed of amino acid residuesspecifically conserved among the MenE family (Fig. 3). Impor-tantly, the two substrates are perfectly positioned for in-linebackside nucleophilic substitution reaction with a short oxy-gen-to-phosphorus distance between the reacting functionalgroups (Fig. 6). This allows us a glimpse of how the enzymesequentially binds the two substrates and aligns them justbefore the reaction happens. It also provides a general model foralignment of the two substrates for the first half-adenylationreaction catalyzed by other ANL enzymes.

In addition to creating a new substrate binding, the ATPbinding also positions conserved amino acid residues for stabi-lization of the transition state. Noticeably, the ATP-boundBsMenE shows for the first time hydrogen bonding interactionof the invariant A10 Lys-471 with the �-phosphoryl group ofthe ATP substrate, strongly supporting the proposed stabiliza-tion of the developing negative charge in forming the transitionstate of the first step adenylation (32). The fact that this strictlyconserved lysine interacts with the �-phosphate of ATP in DltAand ACSM2A shows that its side chain is highly dynamic andflexible. The crucial catalytic role of the invariant A10 lysineresidue is strongly supported by the complete activity loss in theadenylation half-reaction without affecting the thioesterifica-tion half-reaction in several ANL enzymes when it is mutated(8, 13, 20, 66, 67). It is also supported by the hydrogen bondinginteraction of this residue with the �-phosphoryl group in com-plexes of the adenylate-AMP intermediate or analogues withseveral ANL enzymes, including BsMenE (17, 18, 61, 68) andthe use of acetylation of this residue as a regulation control ofacetyl-CoA synthetase in mammals (46). Noticeably, the hydro-gen bond between this invariant A10 lysine residue and theOSB carboxylate group found in the modeled ATP-Mg2��OSB�-BsMenE complex should align the substrates for the reac-tion and put the side chain in the proper position to stabilize thetransition state, although it may reduce the nucleophilicityof the attacking carboxylate. With the transition state stabiliza-tion by the invariant A10 lysine, the reaction between the twoaligned substrates as visualized in the modeled ATP-OSB-BsMenE complex (Fig. 6), should proceed smoothly to form theadenylate intermediate and pyrophosphate.

Noticeably, a structure relaxation mechanism is intrinsicallyinstalled in the compact ATP-configured adenylate-formingconformation for its easy disassembly, which is required afteradenylation to allow transition to the thioester-forming confor-mation in the domain alteration mechanism. When the adeny-lation is complete in the adenylate-forming conformation, thepyrophosphate product is released easily from one end of theATP-binding tunnel, which is very close to the protein surface(Fig. 2C), to conform to the ordered Bi Uni Uni Bi ping-pongmechanism. This product release eliminates the strong interac-tion of the A8 Arg-382 to the �-phosphate of ATP, which con-trols the correct orientation of the C-domain in the conservedATP-dependent configuration of the adenylation active site.Consequently, the interdomain interaction is significantlyweakened, similar to that observed for the loosened AMP-BsMenE structure (Fig. 2B), to reach a state that allows anotherconformational change to accept the coenzyme A substrate and

form a new active site for the second-step thioesterification.Regaining the C-domain flexibility after adenylation is demon-strated by the two different C-domain orientations found in theOSB-AMP-BsMenE structure (61).

In summary, besides being the first substrate of the ANLenzymes, ATP plays a pivotal structural role in configuringthe active adenylate-forming conformation to impact on theadenylation half-reaction in the ordered substrate binding, thein-line alignment of the two substrates for backside nucleo-philic substitution, and stabilization of the transition state. Thisis achieved by its intense interaction with the P-loop and otherstructural elements, which are highly conserved in the ANLfamily. For this conserved mode of ATP binding in configura-tion of the adenylation active site, it is important to note thatthe strongest interaction affecting the C-domain orientationoccurs at the �-phosphate of the bound ATP. This mode ofC-domain control allows easy disassembly of the adenylate-forming conformation to release the C-domain for a large anglerotation in configuration of the second active site for thioesteri-fication, once the first half-reaction releases its pyrophosphateproduct to eliminate the domain-affecting interaction. Conse-quently, the ATP configuration of the adenylate-forming con-formation is transient and is intimately coupled to progressionof the adenylation half-reaction. Revelation of this structuralrole of ATP has provided novel mechanistic insights into theadenylation half-reaction and the transition to the thioesterifi-cation half-reaction to allow us a better understanding of thedomain alteration mechanism of the adenylating enzymes inthe ANL family.

Author Contributions—Z. G. conceived and coordinated the study.Z. G. and Y. C. wrote the paper. Y. C. designed, performed, and ana-lyzed experiments in all figures. Y. S. and H. S. participated in proteinpurification, protein crystallization, data collection, and structuraldetermination. All authors reviewed the results and approved thefinal version of the manuscript.

Acknowledgments—We are grateful for access to beamline BL17U1 atShanghai Synchrotron Radiation Facility (SSRF), and we thank thebeamline staff for technical support.

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Yaozong Chen, Yueru Sun, Haigang Song and Zhihong Guo-Succinylbenzoyl-CoA SynthetaseBacillus subtilis oin

Structural Basis for the ATP-dependent Configuration of Adenylation Active Site

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