Neutron structures of the Helicobacter pylori5′-methylthioadenosine nucleosidase highlightproton sharing and protonation statesMichael T. Bancoa, Vidhi Mishraa, Andreas Ostermannb, Tobias E. Schraderc, Gary B. Evansd, Andrey Kovalevskye,and Donald R. Ronninga,1

aDepartment of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606; bHeinz Maier-Leibnitz Zentrum, Technische Universität München,85748 Garching, Germany; cJülich Centre for Neutron Science at Heinz Maier-Leibnitz Zentrum, Forschungszentrum Jülich GmbH, 85747 Garching,Germany; dFerrier Research Institute, Victoria University of Wellington, Wellington 5010, New Zealand; and eBiology and Soft Matter Division, Oak RidgeNational Laboratory, Oak Ridge, TN 37831

Edited by Dagmar Ringe, Brandeis University, Waltham, MA, and accepted by Editorial Board Member David W. Russell October 21, 2016 (received for reviewJune 16, 2016)

MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydro-lysis of the N-ribosidic bond of a variety of adenosine-containingmetabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes6-amino-6-deoxyfutalosine in the second step of the alternativemenaquinone biosynthetic pathway. Substrate binding of the ad-enine moiety is mediated almost exclusively by hydrogen bonds,and the proposed catalytic mechanism requires multiple proton-transfer events. Of particular interest is the protonation state ofresidue D198, which possesses a pKa above 8 and functions as ageneral acid to initiate the enzymatic reaction. In this study wepresent three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), For-mycin A (FMA), and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA)as well as one neutron/X-ray crystal structure of an inactive variant(HpMTAN-D198N) cocrystallized with SAH. These results support amechanism of D198 pKa elevation through the unexpected sharingof a proton with atom N7 of the adenine moiety possessing uncon-ventional hydrogen-bond geometry. Additionally, the neutron struc-tures also highlight active site features that promote the stabilizationof the transition state and slight variations in these interactions thatresult in 100-fold difference in binding affinities between theDADMe-ImmA and ImmA analogs.

neutron diffraction | enzyme mechanism | proton transfer | nucleosidase |Helicobacter

The Gram-negative bacterium Helicobacter pylori is associatedwith gastric ulcers as well as chronic gastritis. Menaquinone(vitamin K2) is an essential metabolite that aids in electron transferin all organisms. In contrast to most bacteria that use the classicalmenaquinone biosynthetic pathway, H. pylori and Campylobacterjejuni use what is now termed the “alternative” menaquinonebiosynthetic pathway to produce menaquinone from chorismate(1). Therefore enzymes that function within this pathway are at-tractive candidates for developing H. pylori-specific treatments.One such target in this pathway is a homodimeric enzyme, H. pylori5′-methylthioadenosine nucleosidase (HpMTAN), that hydrolyzesthe N-ribosidic bond of 6-amino-6-deoxyfutalosine (Fig. 1A) (2–4).Additionally, HpMTAN hydrolyzes the N-ribosidic bond of otheradenosine-containing metabolites such as S-adenosylhomocysteine(SAH) (Table S1) and 5′-deoxyadenosine (5–7) and thereforefunctions as a central metabolic hub.The proposed catalytic reaction of HpMTAN progresses through

an SN1 mechanism and has been well studied for various MTANhomologs (Fig. 1B) (8–13). Catalysis is initiated by protonationof N7 of the adenine moiety by an aspartic acid residue, D198.Maintaining the protonated form of D198 requires elevation ofthe side chain pKa to a level much higher than the theoretical pKaof an aspartic acid side chain. Indeed, assessment of side-chain

ionization of the analogous residue in the Escherichia coli homologdetermined a pKa of 8.2, which can be attributed to the burial ofthe D198 side chain upon substrate binding (9, 14). Additionally,D198 has been demonstrated to be essential for the enzymaticactivity through the use of an asparagine variant (D198N) ofHpMTAN that binds substrate but does not promote hydrolysis (5,12, 15). Following protonation of the substrate by D198, the ade-nine leaving group becomes electron withdrawing, leading toelongation of the N-ribosidic bond. This elongation promotes bondbreakage, producing an oxocarbenium ion intermediate. A boundwater molecule in the active site functions as a nucleophile to at-tack the oxocarbenium ion intermediate. In previous studies ofE. coliMTAN (EcMTAN), conserved residues E12 and E175 wereshown to be essential for the catalytic reaction based on inactivevariants (6). Furthermore, it was suggested that E12 functions as ageneral base by activating the nucleophilic water molecule, becausea second ionizable group with a pKa value of 5.6 was identified (9).In the various MTAN homologs, it has been shown that the

formation of the oxocarbenium ion intermediate can progressthrough either an early or late dissociative transition state. Thestructure of the transition state is defined by the distance from theN9 position of the adenine leaving group to the anomeric carbon onthe ribosyl moiety of a substrate. Characterization of the HpMTANtransition state has been demonstrated previously to be an earlydissociative transition state by measuring kinetic isotope effectsusing both Immucillin-A (ImmA) and DADMe-Immucillin-A

Significance

Gastrointestinal infection by the bacterium Helicobacter pylori isstrongly associated with the development of gastric cancer.H. pylori 5′-methylthioadenosine nucleosidase (HpMTAN) isan interesting drug target because of its vital role in the pro-duction of menaquinone. HpMTAN offers a unique target fortreating H. pylori infections without affecting the survival of thehuman microbiome. Neutron crystallography was performed todetermine hydrogen atom positions that provide insight into thecatalytic mechanism and transition state stabilization.

Author contributions: D.R.R. designed research; M.T.B., V.M., A.O., T.E.S., and A.K. per-formed research; G.B.E. contributed new reagents/analytic tools; M.T.B. and D.R.R. ana-lyzed data; and M.T.B. and D.R.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. D.R. is a Guest Editor invited by the EditorialBoard.

Data deposition: Crystallography, atomic coordinates, and structure factors reported inthis paper have been deposited in the Protein Data Bank (PDB ID codes 5CCD, 5K1Z, 5JPC,5CCE, and 5KB3).1To whom correspondence should be addressed. Email: donald.ronning@utoledo.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1609718113/-/DCSupplemental.

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(DADMe-ImmA) derivatives (16). These compounds are pro-tonated at the N7 position of the adenine moiety but differ sig-nificantly in the ribose mimic and vary in the distance between theribose- and adenine-mimicking moieties. The ImmA early disso-ciative transition state analogs feature a cationic 4′ iminoribitolnitrogen atom; the distance between the nonhydrolyzable adeninemimic and the C1′ of the iminoribitol is 1.4 Å. The DADMe-ImmAanalogs represent a late dissociative transition state by containing acationic nitrogen atom in the pyrrolidine moiety that represents afully formed carbocation as well as having an elongated distance of2.5 Å between the nonhydrolyzable adenine mimic and pyrrolidinemoiety. Although previously published kinetic data identifyHpMTAN as forming an early dissociative transition state duringcatalysis, the most potent transition-state analogs are DADMe-ImmA derivatives, which exhibit Kd values in the picomolarrange (16, 17).Determination of the protonation states of specific residues

and the positioning of hydrogen atoms are important for un-derstanding the catalytic mechanism as well as for substrate andinhibitor recognition. Protons are readily observed in moderate-resolution neutron crystallographic studies (18). Here we presentnucleosidase neutron crystal structures. The enzyme was cocrystal-lized with four ligands that represent a Michaelis complex, bothearly and fully dissociative transition state complexes, and a productcomplex. The binary Michaelis complex mimic (HpMTAN-D198N/SAH) neutron structure was solved to 2.6-Å resolution. The D198Nvariant inactivates the enzyme but allows substrate binding. Addi-tionally, neutron crystal structures of wild-type HpMTAN in com-plex with two transition-state analogs, Formycin A (FMA;HpMTAN/FMA) and (3R,4S)-4-(4-Chlorophenylthiomethyl)-1-[(9-deaza-adenin-9-yl)methyl]-3-hydroxypyrrolidine (p-ClPh-Thio-DADMe-ImmA; HpMTAN/p-ClPh-Thio-DADMe-ImmA),were solved to 2.5 Å and 2.6 Å, respectively, providing structural

snapshots of the active site of HpMTAN when stabilizing eitherthe early (FMA) or late (DADMe-ImmA) dissociative transitionstates (19). Last, the neutron structure of the product complex[HpMTAN/S-ribosylhomocysteine (SRH)/adenine] was solved to2.5-Å resolution. These neutron structures further define the cat-alytic mechanism of HpMTAN by presenting the protonationstates of the several polar groups essential for the enzymatic re-action. Additionally, the observed deuterium positions illustratethe interactions within the enzyme active site that stabilize thetransition state and provide insights into the differences in HpMTANaffinity between FMA and the DADMe-ImmA compounds.

Materials and MethodsNeutron Crystallography. All neutron diffraction data were collected at roomtemperature. Monochromatic neutron diffraction data were collected on theHpMTAN/SRH/adenine complex using the BIODIFF beam line at the FRM IIresearch reactor at the Heinz Maier-Leibnitz Zentrum. The diffraction datawere indexed and integrated using DENZO and then were scaled withSCALEPACK (20). Quasi-Laue neutron diffraction data were collected on thetransition-state analogs and binary substrate complex crystals at roomtemperature on the IMAGINE beamline located at the high-flux isotopereactor (HFIR) at Oak Ridge National Laboratory, Oak Ridge, TN (21). Theneutron data were processed using the Daresbury Laboratory LAUE suiteprogram LAUEGEN modified to account for the cylindrical geometry of thedetector (22, 23). The program LSCALE was used to determine the wave-length-normalization curve using the intensities of symmetry-equivalentreflections measured at different wavelengths and merged in SCALA (24,25). The crystallographic data are given in Table S2.

X-Ray Crystallography. X-ray crystallographic datasets were collected at roomtemperature using crystals taken from the same crystallization drops thatprovided crystals for the neutron diffraction studies. Datasets were collectedon a Rigaku HighFlux HomeLab instrument. The high-resolution X-ray dif-fraction datasetwas collected in cryogenic conditions (95 K) at the LS-CAT ID-Fbeamline at the Advanced Photon Source, Argonne National Laboratory.Diffraction datasets were collected, integrated, and scaled using the HKL3000software suite (26). Structures were solved by the molecular replacementmethod using phasing information from the HpMTAN/FMA structure withProtein Data Bank (PDB) ID code 3NM5 (5). The HpMTAN/SRH/adenineX-ray structure was refined using SHELX-97 (27). The high-resolutionHpMTAN/p-ClPh-Thio-DADMe-ImmA structure and room-temperatureX-ray datasets for the joint X-ray/neutron (XN) refinements were refinedusing PHENIX (28). The crystallographic data for the joint XN- datasetsare given in Table S2, and the high-resolution X-ray dataset is given inTable S3.

Fig. 1. (A) A surface model of HpMTAN in complex with SAH. The enzyme isan obligatory homodimer in which the two chains of the dimer are repre-sented by the white and the green surfaces. The red box highlights theseveral hydrogen-bond interactions that contribute to substrate binding inthe HpMTAN active site. (B) A structural representation of the HpMTANcatalytic reaction (PDB ID codes 4OY3 and 4OTJ). (Left) Substrate and thenucleophilic water-bound molecule. After formation of the oxocarbeniumion intermediate, a bound water molecule in the active site attacks the C1′of the ribosyl moiety, resulting in the hydrolysis of the substrate. (Right) Theproduct complex.

Fig. 2. The observed positions of the deuterium atom in the adenine-binding pocket of HpMTAN for the product complex presented. In bothpanels, the 2FO-FC for the nuclear density is contoured to 1σ and is repre-sented in light blue. The FO-FC nuclear and X-ray density are represented indark blue and light green, respectively. (A) The 2FO-FC density illustrating thehydrogen-bond network that aids in positioning D198. (B) Difference omitFO-FC nuclear density of the shared D

+ ion and of the N9 nitrogen anddeuteron atoms contoured to 3.5σ. The FO-FC X-ray map of the adeninemolecule and D198, contoured to 3.0σ, demonstrates the lack of density forthe shared D+ ion. Hydrogen-bonding interactions and the respective dis-tances are indicated.

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Joint XN Refinement. The jointly refined XN structures were all refined usingnCNS (29). After initial rigid-body refinement, several cycles of positional,atomic displacement parameter, and occupancy refinement followed.Within each refinement cycle the structure was manually checked using Coot(30). The 2FO-FC and FO-FC neutron scattering length density maps then wereexamined to determine the correct orientation and protonation states ofresidues with exchangeable protons. All water molecules were refined asD2O. Initially, water oxygen atoms were positioned using X-ray differencemaps and then were shifted slightly in accordance with the neutron scat-tering length density maps. The levels of hydrogen/deuterium (H/D) ex-change at OH, NH, and SH sites were refined using D occupancy as themetric. All structures have been deposited in the Protein Data Bank (PDB IDcodes 5CCD, 5K1Z, 5JPC, 5CCE, and 5KB3).

Results and DiscussionDefining Proton Positions During the Catalytic Reaction of HpMTAN.It is well accepted that the initiation of the catalytic reaction ofHpMTAN is afforded by the donation of a proton from D198 tothe N7 position of the substrate. The positioning of D198 de-scribed in the transition-state analogs and product complexes isafforded by three hydrogen-bond interactions from the hydroxylside chain of S197, the backbone amide of A200, and N6 of theadenine moiety (Fig. 2A). The deprotonated adenine N7 nitro-gen atom has been calculated to possess negative electrostaticpotential before protonation, promoting interaction with andproton transfer from D198 (19). Inspection of the omit differ-ence FO-FC nuclear map of the product complex reveals strongdifference density for a shared deuterium ion between the car-boxylic acid moiety of D198 and N7 of adenine (Fig. 2B). Thepresence of the shared D+ ion was unexpected because it has notbeen previously proposed. The D+ ion develops a trifurcatedhydrogen bond that is positioned at a distance of 1.8 Å and 2.3 Åfrom Oδ1 and Oδ2 of D198, respectively, and 1.7 Å from N7 of theadenine. The angle between N7···D+···Oδ1 is 98° and betweenN7···D+···Oδ2 is 116°, deviating significantly from the more common180° in countless biological interactions or the 155° angle observed inthe MTAN transition-state analog neutron structures. Interestingly,the bonding distances of N···D+ and D+···Oδ1 are nearly equivalent,suggesting that the pKa values of D198 and N7 are closely matchedand implying a possible low-barrier hydrogen bond (31). Visualiza-tion of the shared D+ ion further confirms the involvement of D198in the initiation of the catalytic reaction by protonation of the ade-nine N7. In addition, this hydrogen-bond network elevates the pKa ofthe D198 side chain and ensures that a proton is available in theactive site for subsequent enzyme-catalyzed reactions.Additionally, omit difference FO-FC nuclear density was observed

for a deuterium atom on the N9 position of the adenine molecule,further highlighting the importance of the shared D+ ion in the

catalytic mechanism (Fig. 2B). In an accepted mechanism forMTAN, after the initiation of the catalytic reaction, the presence ofthe resulting carboxylate moiety of D198 develops an N7-H···Oδ1hydrogen-bond interaction with the protonated N7 of the substrate.The transient positive charge of the adenine moiety consequentlyleads to breakage of the N-ribosidic bond, resulting in an adeninemolecule with a protonated N7 and an unprotonated N9 (12, 32).This conformation directly conflicts with the protonation statesobserved in the product complex and suggests that, after disruptionof the N-ribosidic bond, the N9 accepts a proton, and N7 beginssharing its proton with the D198 side chain instead of retaining theproton. An intriguing question addressed by the protonation statesof the adenine product is the identity of the specific chemical groupthat donates a proton to N9. It has been proposed that the con-served residue E13 acts as a general base to activate the nucleo-philic water molecule that attacks the intermediate during theenzymatic reaction (9, 12, 33). Examination of the structure of theternary product complex shows that the protonated N9 is 6.7 Åfrom E13 and lacks neighboring proton acceptors that could allowa proton shuttle-like mechanism. Additionally, all the neutronstructures presented here show E13 to be in the deprotonated,carboxylate form. Therefore, it is unlikely the observed pKa of 5.6is the result of E13 functioning as a general base in activating thenucleophile but instead ensures a fully deprotonated carboxylateto orient the nucleophilic water molecule properly for attack onthe oxocarbenium ion intermediate. The nucleophilic water mol-ecule then directly donates a proton to N9 subsequent to nucle-ophilic attack on the oxocarbenium ion intermediate. Theseobservations of the deuterium positions of the subsequent adenineproduct better define the proton transfer events involved in ca-talysis (Fig. 3) (34).The SRH-binding subsite of the HpMTAN active site contains

a variety of polar residues that allow the recognition of the ribosemoiety of the substrate, some of which have ambiguous hydro-gen-bond donors and acceptors. In the substrate complex, theobserved nucleophilic water molecule is positioned by hydrogen-bond interactions with both the guanidinium moiety of R194 anda fully deprotonated carboxylate moiety of E13 with distances of1.8 Å and 1.6 Å, respectively (Fig. 4). The orientation of theputative nucleophile provided by these residues ensures the po-sitioning of a lone pair of electrons in the nucleophile toward theanomeric carbon of the ribose moiety before the formation ofthe oxocarbenium ion intermediate. Following nucleophilic at-tack by the water molecule on the intermediate, a fully depro-tonated E13 is observed forming a hydrogen-bond interactionwith the O1′ hydroxyl of SRH in the product complex. Thisconserved glutamate residue has been demonstrated to be

Fig. 3. The proposed catalytic mechanism of HpMTAN. The colored hydrogen atoms represent the protons that are involved in the enzymatic reactiondetermined by the neutron structures. Briefly, the catalytic reaction is initiated by D198 protonating the N7 of the adenyl moiety. Delocalization of electronsin the adenyl moiety leads to bond elongation of the N-ribosidic bond, consequently forming an oxocarbenium ion intermediate. A bound water moleculethen will attack the C1′ of the ribosyl moiety and will donate a proton to the N9 of adenine after the N7 proton is shared.

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important for the catalytic reaction based on a significant loss ofactivity in EcMTAN variants E12Q (EcMTAN-E12Q) and E12A(6). The neutron structures described here offer an interestingalternative hypothesis for the significant, but not complete, lossof enzymatic activity in the EcMTAN variants. Inspection ofeach neutron structure highlights E13 in HpMTAN as a hydro-gen-bond acceptor to the backbone amides of residue A9, M10,and V78 in addition to the nucleophilic water molecule. Theanalogous Q12 residue in EcMTAN-E12Q could alter the hy-drogen-bond network for the nucleophilic water molecule andreorient it within the active site. Because of this improper ori-entation of the nucleophilic water molecule, the lone pair ofelectrons on the oxygen atom of the nucleophile needed to formthe new bond with the C1′ of ribose is oriented toward the Q12side chain in the E12Q variant and away from the oxocarbeniumion intermediate, consequently decreasing the likelihood of nu-cleophilic attack. This hypothesis also can explain why theEcMTAN variants retain a low level of enzymatic activity with-out a general base at this position. Specifically, thermal motionor random reorientation of the nucleophilic water moleculewithin the active site immediately following oxocarbenium ionformation in the EcMTAN-E12Q variant could allow a minorityof the Michaelis complexes to undergo nucleophilic attack andcomplete catalysis.Inspection of the neutron structure for HpMTAN-D198N/

SAH provides additional information that supports an expandedrole of S197 in the catalytic mechanism of HpMTAN. The in-volvement of S197 in the catalytic reaction has been studiedpreviously for EcMTAN with an MTAN-S197A variant thatpossessed only 10% of the wild-type activity (6). Furthermore,S197 was suggested to be the proton donor for D198, allowing

the hydroxyl of S197 to be regenerated by the bulk solventthrough the classical Grotthuss mechanism (13). In the prod-uct complex, S197 and D198 develop a strong, linear, 1.9 Å,O-D···Oδ1 hydrogen-bond interaction (Fig. 5A). Although thisinteraction assists in positioning the D198 side chain to promoteproton donation to the substrate, the presented neutron struc-ture as well as published X-ray structures suggest that S197 mayplay a role in proton transfer to D198 and the substrate. In theHpMTAN-D198N/SAH neutron structure, the deuterium posi-tion on the S197 hydroxyl is reoriented, creating a new hydrogen-bond network involving a water molecule in a small hydrophobicpocket abutting the active site (Fig. 5A). The S197 O-D grouprotates nearly 180°, positioning the deuterium away from the N198side chain as the result of the δND2 group developing an N-D···Ohydrogen bond with a distance of 2.4 Å. The other δND2 atom ofN198 forms an N-D···N hydrogen bond with N7 of the SAH at adistance of 2.1 Å. In the previously published HpMTAN-D198NX-ray crystal structures, this pocket always contains a single or-dered water molecule, but no corresponding density is observedin the wild-type structures (5, 12, 15). Although ordering of thiswater molecule could simply be a consequence of the D198Nmutation, it is expected that this pocket contains a disorderedwater molecule in the wild-type enzyme. The water in the hy-drophobic pocket interacts with S197 by accepting a hydrogenbond from the S197 side chain at a distance of 1.7 Å (Fig. 5B).Additionally, the water molecule within this small hydrophobicpocket develops a hydrogen bond with the backbone carbonylof F208 as well as forming an O-H···π interaction with the sidechain of the highly conserved F208 (35, 36). The hydrogen-bonding pattern suggests a possible mechanism for the bindingof a hydronium ion within this pocket and a proton shuttle toD198 mediated by S197. Whether this water molecule plays arole in the enzyme mechanism or is simply a function of theinactivating D198N mutation is unclear. It is intriguing, how-ever, to consider that the binding of a hydronium ion to this sitein the wild-type enzyme could be stabilized through a cation-πinteraction with F208 and promote D198 protonation via aproton shuttle through S197. Such a role for ordered watermolecules has been highlighted in other neutron structures(37). This scenario offers a possible second mechanism bywhich the enzyme ensures a protonated D198 residue uponbinding substrate.

Insights into Transition-State Stabilization and Inhibitor Affinity. Inthe transition-state analog neutron structures, specifically FMAand p-ClPh-Thio-DADMe-ImmA, the observed deuterium

Fig. 4. Positioning of the nucleophilic water molecule in the substratecomplex. The nuclear and X-ray 2Fo-Fc maps are contoured to 1σ. The nu-clear 2FO-FC map is presented in light blue, and the X-ray 2FO-FC map ispresented in green. The positioning of the nucleophilic water molecule isafforded by hydrogen-bonding interactions with the carboxylate moiety ofE13 and the guanidinium moiety of R194. The hydrogen-bonding interac-tions with the two residues allow one lone pair of electrons of the watermolecule to be positioned near the oxocarbenium ion intermediate to allownucleophilic attack at the C1′ position of the ribose moiety as indicated bythe arrow.

Fig. 5. The observed positions of deuterium atoms in the adenine-bindingpocket of HpMTAN-D198N for the inactive binary substrate complex. In bothpanels, the 2FO-FC for the nuclear and X-ray density is contoured to 1σ and isrepresented in light blue and green, respectively. (A) Due to the D198Nvariant, the hydrogen of the hydroxyl group of S197 is reoriented to interactwith a bound water molecule. (B) The interactions between the watermolecule and the conserved F208 residue.

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positions provide information about the specific hydrogen-bondinteractions that contribute to the high-affinity binding of theseinhibitors and to stabilization of the transition state. Differ-ences in the hydrogen-bond interactions of the two transition-state analogs were observed in the ribose-binding site, whereasthe hydrogen-bond interactions in the adenine-binding pocketwere conserved in both transition state complexes. In theHpMTAN/FMA and HpMTAN/p-ClPh-Thio-DADMe-ImmAneutron structures, 2FO-FC nuclear density indicates a pro-tonated N7 atom mimicking the protonated substrate immedi-ately after the initiation of the catalytic reaction. The stronghydrogen-bond interaction between the deprotonated carboxyl-ate group of D198 and the N7 proton of the nonhydrolyzableadenine mimics retains a distance of 2.0 Å and an angle of155° between the three atoms in both complexes. Interestingly,slight variations in the hydrogen-bond distance were observedbetween the hydroxyl of S197 and the carboxylate of D198 inboth transition-state analog structures as compared with theproduct complex. In the p-ClPh-Thio-DADMe-ImmA complex,the observed O-D···Oδ1 hydrogen-bond distance increased to2.4 Å with a 142° angle. The FMA complex demonstrated amore dramatic change in the O-D···Oδ1 hydrogen bond con-sisting of 2.6 Å with 112° angles between the three atoms. Inboth the p-ClPh-Thio-DADMe-ImmA and the product com-plex, the hydroxyl of S197 forms a van der Waals interactionwith the C8 hydrogen atom of adenine, which in FMA isreplaced by a nitrogen atom. Therefore, the loss of the vander Waals interaction between the S197 hydroxyl and thesubstrate weakens the hydrogen bond interaction betweenS197 and D198.The HpMTAN/p-ClPh-Thio-DADMe-ImmA complex dem-

onstrates several hydrogen-bond interactions that contribute tothe picomolar affinity of this inhibitor even though HpMTAN issuggested to form an early dissociative transition state. It wasshown that the Kd for p-ClPh-Thio-DADMe-ImmA was 570 pMfor HpMTAN, an affinity roughly 100-fold higher than that ofthe ImmA analog that was demonstrated to have a Kd of 40 nM(16). The differences in the affinities of the transition state an-alogs to structurally similar homologs of MTAN have beenstudied previously using computational techniques (38, 39).2FO-FC nuclear density for the HpMTAN/p-ClPh-Thio-DADMe-ImmA complex demonstrates a deuterium atom located on theN1′ of the pyrrolidine moiety, confirming the presence of thecationic nitrogen atom in the catalytic site of HpMTAN (Fig.6A). Therefore, this neutron structure gives a direct view of thehydrogen-bond interactions that orient a lone pair of electronson the nucleophilic water molecule toward the newly formedoxocarbenium ion intermediate. The oxygen atom of the nucle-ophile is observed to form a strong N-D···O hydrogen-bondinginteraction from both the guanidinium moiety of R194 and thecationic nitrogen atom of the N1′ from the pyrrolidine moietywith distances of 1.8 Å and 1.6 Å, respectively. Additionally, thedeuterium atoms of the nucleophile form moderate-strengthO-D···O hydrogen-bonding interactions with the deprotonatedcarboxylic acid moieties of E13 and E175 at distances of 2.3 Åand 2.5 Å, respectively. Besides the other various interactionsbetween p-ClPh-Thio-DADMe-ImmA and HpMTAN, the ob-served hydrogen bonded network with the nucleophile contrib-utes significantly to the proper orientation of the water moleculefor attack on the oxocarbenium ion intermediate. To confirmthat the high-affinity binding of p-ClPh-Thio-DADMe-ImmA iscaused by the presence of a cationic character and not by pos-sible flattening of the pyrrolidine ring, a 1.4-Å resolution X-raycrystal structure was refined showing that the N1′ is sp3hybridized (Fig. S1).Inspection of the HpMTAN/FMA complex provides evi-

dence regarding the difference in affinities observed betweenDADMe-ImmA and ImmA transition-state analogs (Fig. 6B).

In the HpMTAN/FMA complex an unusual binding orientationof the nucleophilic water molecule was observed that does notallow a lone pair of electrons to be positioned toward the in-hibitor. This unusual orientation of the water molecule isafforded by strong and moderate hydrogen-bond interactionswith the guanidinium moiety of R194, the carboxylate moietyof E13, and the O3′ hydroxyl of the ribose moiety of FMA.Intriguingly, in the HpMTAN/FMA neutron structure the 2FO-FC nuclear map shows that the deuterium atom of the O3′hydroxyl for the ribose moiety is rotated 76° from the Oδ2 ofE175 toward the 5′-alkylthio–binding subsite of the catalyticsite. Although an increase in the distance between the hydro-gen-bond acceptor and donor was not observed, the new pro-ton position disrupts the hydrogen-bond interaction with Oδ2of E175 that is observed in all other neutron structures. Thenew orientation of the deuterium atom for the O3′ hydroxylallows a new O-D···O hydrogen-bond interaction to form with theO2′ hydroxyl at a distance of 2.1 Å. The O-D···Oδ2 hydrogen-bond interaction between the O3′ hydroxyl and the carboxylateside chain of E175 was shown to be essential for the catalytic re-action, because the removal of the O3′ hydroxyl from the ribosemoiety and the use of E175 EcMTAN variants eliminate catalyticactivity (6, 9). Based on these results, it has been proposed that theO3′ hydroxyl becomes ionized during the formation of the tran-sition state to assist in stabilizing the oxocarbenium ion in-termediate (11, 32), but the neutron structures presented heredemonstrate a protonated O3′ hydroxyl and a fully deprotonatedE175 carboxylate.

ACKNOWLEDGMENTS. We thank Heinz Maier-Leibnitz Zentrum and OakRidge National Laboratory for graciously providing the beam time that wasessential for this work. This work was supported by the Center for theAdvancement of Science in Space via a cooperative agreement with Na-tional Aeronautics and Space Administration Grant N-123528-01 (to D.R.R)and by National Institute of Allergy and Infectious Disease/NIH GrantAI105084 (to D.R.R.). This research used the resources of the AdvancedPhoton Source, a US Department of Energy (DOE) Office of Science UserFacility operated for the DOE Office of Science by Argonne National Lab-oratory under Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21was supported by the Michigan Economic Development Corporation andby Grant 085P1000817 from the Michigan Technology Tri-Corridor. Theresearch was sponsored in part by the Scientific User Facilities Division,Office of Basic Energy Sciences, U.S. Department of Energy. The IMAGINEProject was partially supported by the National Science Foundation (Grant0922719).

Fig. 6. The hydrogen-bonding interactions observed for the nucleophilein the presence of the fully and early dissociative transition-state analogs.The 2FO-FC nuclear and X-ray maps are contoured to 1σ and represented inlight blue and green, respectively. (A) The deuteron positions of the nu-cleophile and the various hydrogen-bond interactions with p-ClPh-Thio-DADMe-ImmA are shown. Additionally, the 2FO-FC nuclear maps show aprotonated ammonium in the pyrrolidine moiety of the DADMe-ImmAanalog. (B) Positioning of the observed deuterium atoms for FMA and theunusual orientation of the nucleophile in the HpMTAN/FMA neutronstructure. In the FMA complex the deuterium atom of the 3′ hydroxyl ofthe ribose moiety is in an unexpected position that disrupts the hydrogen-bond interaction with Oδ2 of E175 that was observed in all the otherneutron structures.

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