Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Michael T. Banco; Vidhi Mishra; Andreas Ostermann; Tobias E. Schrader; Gary B. Evans; Andrey Kovalevsky; Donald R. Ronning

doi:10.1073/pnas.1609718113

New Research In

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

Significance

Gastrointestinal infection by the bacterium Helicobacter pylori is strongly associated with the development of gastric cancer. H. pylori 5′-methylthioadenosine nucleosidase (HpMTAN) is an interesting drug target because of its vital role in the production of menaquinone. HpMTAN offers a unique target for treating H. pylori infections without affecting the survival of the human microbiome. Neutron crystallography was performed to determine hydrogen atom positions that provide insight into the catalytic mechanism and transition state stabilization.

Abstract

MTAN (5′-methylthioadenosine nucleosidase) catalyzes the hydrolysis of the N-ribosidic bond of a variety of adenosine-containing metabolites. The Helicobacter pylori MTAN (HpMTAN) hydrolyzes 6-amino-6-deoxyfutalosine in the second step of the alternative menaquinone biosynthetic pathway. Substrate binding of the adenine 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 of residue D198, which possesses a pK_a above 8 and functions as a general acid to initiate the enzymatic reaction. In this study we present three corefined neutron/X-ray crystal structures of wild-type HpMTAN cocrystallized with S-adenosylhomocysteine (SAH), Formycin 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 a mechanism of D198 pKa elevation through the unexpected sharing of a proton with atom N7 of the adenine moiety possessing unconventional hydrogen-bond geometry. Additionally, the neutron structures also highlight active site features that promote the stabilization of the transition state and slight variations in these interactions that result in 100-fold difference in binding affinities between the DADMe-ImmA and ImmA analogs.

The Gram-negative bacterium Helicobacter pylori is associated with gastric ulcers as well as chronic gastritis. Menaquinone (vitamin K₂) is an essential metabolite that aids in electron transfer in all organisms. In contrast to most bacteria that use the classical menaquinone biosynthetic pathway, H. pylori and Campylobacter jejuni use what is now termed the “alternative” menaquinone biosynthetic pathway to produce menaquinone from chorismate (1). Therefore enzymes that function within this pathway are attractive candidates for developing H. pylori-specific treatments. One such target in this pathway is a homodimeric enzyme, H. pylori 5′-methylthioadenosine nucleosidase (HpMTAN), that hydrolyzes the N-ribosidic bond of 6-amino-6-deoxyfutalosine (Fig. 1A) (2 ⇓–4). Additionally, HpMTAN hydrolyzes the N-ribosidic bond of other adenosine-containing metabolites such as S-adenosylhomocysteine (SAH) (Table S1) and 5′-deoxyadenosine (5 ⇓–7) and therefore functions as a central metabolic hub.

Fig. 1.

(A) A surface model of HpMTAN in complex with SAH. The enzyme is an obligatory homodimer in which the two chains of the dimer are represented by the white and the green surfaces. The red box highlights the several hydrogen-bond interactions that contribute to substrate binding in the HpMTAN active site. (B) A structural representation of the HpMTAN catalytic reaction (PDB ID codes 4OY3 and 4OTJ). (Left) Substrate and the nucleophilic water-bound molecule. After formation of the oxocarbenium ion intermediate, a bound water molecule in the active site attacks the C1′ of the ribosyl moiety, resulting in the hydrolysis of the substrate. (Right) The product complex.

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Table S1.

Acronyms and chemical structures of compounds

The proposed catalytic reaction of HpMTAN progresses through an S_N1 mechanism and has been well studied for various MTAN homologs (Fig. 1B) (8 ⇓⇓⇓⇓–13). Catalysis is initiated by protonation of N7 of the adenine moiety by an aspartic acid residue, D198. Maintaining the protonated form of D198 requires elevation of the side chain pK_a to a level much higher than the theoretical pK_a of an aspartic acid side chain. Indeed, assessment of side-chain ionization of the analogous residue in the Escherichia coli homolog determined a pK_a of 8.2, which can be attributed to the burial of the D198 side chain upon substrate binding (9, 14). Additionally, D198 has been demonstrated to be essential for the enzymatic activity through the use of an asparagine variant (D198N) of HpMTAN that binds substrate but does not promote hydrolysis (5, 12, 15). Following protonation of the substrate by D198, the adenine leaving group becomes electron withdrawing, leading to elongation of the N-ribosidic bond. This elongation promotes bond breakage, producing an oxocarbenium ion intermediate. A bound water molecule in the active site functions as a nucleophile to attack the oxocarbenium ion intermediate. In previous studies of E. coli MTAN (EcMTAN), conserved residues E12 and E175 were shown to be essential for the catalytic reaction based on inactive variants (6). Furthermore, it was suggested that E12 functions as a general base by activating the nucleophilic water molecule, because a second ionizable group with a pK_a 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 progress through either an early or late dissociative transition state. The structure of the transition state is defined by the distance from the N9 position of the adenine leaving group to the anomeric carbon on the ribosyl moiety of a substrate. Characterization of the HpMTAN transition state has been demonstrated previously to be an early dissociative transition state by measuring kinetic isotope effects using both Immucillin-A (ImmA) and DADMe-Immucillin-A (DADMe-ImmA) derivatives (16). These compounds are protonated at the N7 position of the adenine moiety but differ significantly in the ribose mimic and vary in the distance between the ribose- and adenine-mimicking moieties. The ImmA early dissociative transition state analogs feature a cationic 4′ iminoribitol nitrogen atom; the distance between the nonhydrolyzable adenine mimic and the C1′ of the iminoribitol is 1.4 Å. The DADMe-ImmA analogs represent a late dissociative transition state by containing a cationic nitrogen atom in the pyrrolidine moiety that represents a fully formed carbocation as well as having an elongated distance of 2.5 Å between the nonhydrolyzable adenine mimic and pyrrolidine moiety. Although previously published kinetic data identify HpMTAN as forming an early dissociative transition state during catalysis, the most potent transition-state analogs are DADMe-ImmA derivatives, which exhibit K_d values in the picomolar range (16, 17).

Determination of the protonation states of specific residues and the positioning of hydrogen atoms are important for understanding the catalytic mechanism as well as for substrate and inhibitor recognition. Protons are readily observed in moderate-resolution neutron crystallographic studies (18). Here we present nucleosidase neutron crystal structures. The enzyme was cocrystallized with four ligands that represent a Michaelis complex, both early and fully dissociative transition state complexes, and a product complex. The binary Michaelis complex mimic (HpMTAN-D198N/SAH) neutron structure was solved to 2.6-Å resolution. The D198N variant inactivates the enzyme but allows substrate binding. Additionally, neutron crystal structures of wild-type HpMTAN in complex 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 either the early (FMA) or late (DADMe-ImmA) dissociative transition states (19). Last, the neutron structure of the product complex [HpMTAN/S-ribosylhomocysteine (SRH)/adenine] was solved to 2.5-Å resolution. These neutron structures further define the catalytic mechanism of HpMTAN by presenting the protonation states of the several polar groups essential for the enzymatic reaction. Additionally, the observed deuterium positions illustrate the interactions within the enzyme active site that stabilize the transition state and provide insights into the differences in HpMTAN affinity between FMA and the DADMe-ImmA compounds.

Materials and Methods

Neutron Crystallography.

All neutron diffraction data were collected at room temperature. Monochromatic neutron diffraction data were collected on the HpMTAN/SRH/adenine complex using the BIODIFF beam line at the FRM II research reactor at the Heinz Maier-Leibnitz Zentrum. The diffraction data were indexed and integrated using DENZO and then were scaled with SCALEPACK (20). Quasi-Laue neutron diffraction data were collected on the transition-state analogs and binary substrate complex crystals at room temperature on the IMAGINE beamline located at the high-flux isotope reactor (HFIR) at Oak Ridge National Laboratory, Oak Ridge, TN (21). The neutron data were processed using the Daresbury Laboratory LAUE suite program LAUEGEN modified to account for the cylindrical geometry of the detector (22, 23). The program LSCALE was used to determine the wavelength-normalization curve using the intensities of symmetry-equivalent reflections measured at different wavelengths and merged in SCALA (24, 25). The crystallographic data are given in Table S2.

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Table S2.

Diffraction data and refinement statistics for joint XN structures

X-Ray Crystallography.

X-ray crystallographic datasets were collected at room temperature using crystals taken from the same crystallization drops that provided crystals for the neutron diffraction studies. Datasets were collected on a Rigaku HighFlux HomeLab instrument. The high-resolution X-ray diffraction dataset was collected in cryogenic conditions (95 K) at the LS-CAT ID-F beamline at the Advanced Photon Source, Argonne National Laboratory. Diffraction datasets were collected, integrated, and scaled using the HKL3000 software suite (26). Structures were solved by the molecular replacement method using phasing information from the HpMTAN/FMA structure with Protein Data Bank (PDB) ID code 3NM5 (5). The HpMTAN/SRH/adenine X-ray structure was refined using SHELX-97 (27). The high-resolution HpMTAN/p-ClPh-Thio-DADMe-ImmA structure and room-temperature X-ray datasets for the joint X-ray/neutron (XN) refinements were refined using PHENIX (28). The crystallographic data for the joint XN- datasets are given in Table S2, and the high-resolution X-ray dataset is given in Table S3.

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Table S3.

Diffraction data and refinement statistics for high-resolution HpMTAN/p-ClPh-Thio-DADMe-ImmA X-ray structure

Joint XN Refinement.

The jointly refined XN structures were all refined using nCNS (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 2F_O-F_C and F_O-F_C neutron scattering length density maps then were examined to determine the correct orientation and protonation states of residues with exchangeable protons. All water molecules were refined as D₂O. Initially, water oxygen atoms were positioned using X-ray difference maps and then were shifted slightly in accordance with the neutron scattering length density maps. The levels of hydrogen/deuterium (H/D) exchange at OH, NH, and SH sites were refined using D occupancy as the metric. All structures have been deposited in the Protein Data Bank (PDB ID codes 5CCD, 5K1Z, 5JPC, 5CCE, and 5KB3).

Results and Discussion

Defining Proton Positions During the Catalytic Reaction of HpMTAN.

It is well accepted that the initiation of the catalytic reaction of HpMTAN is afforded by the donation of a proton from D198 to the N7 position of the substrate. The positioning of D198 described in the transition-state analogs and product complexes is afforded by three hydrogen-bond interactions from the hydroxyl side chain of S197, the backbone amide of A200, and N6 of the adenine moiety (Fig. 2A). The deprotonated adenine N7 nitrogen atom has been calculated to possess negative electrostatic potential before protonation, promoting interaction with and proton transfer from D198 (19). Inspection of the omit difference F_O-F_C nuclear map of the product complex reveals strong difference density for a shared deuterium ion between the carboxylic acid moiety of D198 and N7 of adenine (Fig. 2B). The presence of the shared D⁺ ion was unexpected because it has not been previously proposed. The D⁺ ion develops a trifurcated hydrogen bond that is positioned at a distance of 1.8 Å and 2.3 Å from Oδ1 and Oδ2 of D198, respectively, and 1.7 Å from N7 of the adenine. The angle between N7⋅⋅⋅D⁺⋅⋅⋅Oδ1 is 98° and between N7⋅⋅⋅D⁺⋅⋅⋅Oδ2 is 116°, deviating significantly from the more common 180° in countless biological interactions or the 155° angle observed in the MTAN transition-state analog neutron structures. Interestingly, the bonding distances of N⋅⋅⋅D⁺ and D⁺⋅⋅⋅Oδ1 are nearly equivalent, suggesting that the pK_a values of D198 and N7 are closely matched and implying a possible low-barrier hydrogen bond (31). Visualization of the shared D⁺ ion further confirms the involvement of D198 in the initiation of the catalytic reaction by protonation of the adenine N7. In addition, this hydrogen-bond network elevates the pK_a of the D198 side chain and ensures that a proton is available in the active site for subsequent enzyme-catalyzed reactions.

Fig. 2.

The observed positions of the deuterium atom in the adenine-binding pocket of HpMTAN for the product complex presented. In both panels, the 2F_O-F_C for the nuclear density is contoured to 1σ and is represented in light blue. The F_O-F_C nuclear and X-ray density are represented in dark blue and light green, respectively. (A) The 2F_O-F_C density illustrating the hydrogen-bond network that aids in positioning D198. (B) Difference omit F_O-F_C nuclear density of the shared D⁺ ion and of the N9 nitrogen and deuteron atoms contoured to 3.5σ. The F_O-F_C X-ray map of the adenine molecule and D198, contoured to 3.0σ, demonstrates the lack of density for the shared D⁺ ion. Hydrogen-bonding interactions and the respective distances are indicated.

Additionally, omit difference F_O-F_C 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 for MTAN, after the initiation of the catalytic reaction, the presence of the resulting carboxylate moiety of D198 develops an N7-H⋅⋅⋅Oδ1 hydrogen-bond interaction with the protonated N7 of the substrate. The transient positive charge of the adenine moiety consequently leads to breakage of the N-ribosidic bond, resulting in an adenine molecule with a protonated N7 and an unprotonated N9 (12, 32). This conformation directly conflicts with the protonation states observed in the product complex and suggests that, after disruption of the N-ribosidic bond, the N9 accepts a proton, and N7 begins sharing its proton with the D198 side chain instead of retaining the proton. An intriguing question addressed by the protonation states of the adenine product is the identity of the specific chemical group that donates a proton to N9. It has been proposed that the conserved residue E13 acts as a general base to activate the nucleophilic water molecule that attacks the intermediate during the enzymatic reaction (9, 12, 33). Examination of the structure of the ternary product complex shows that the protonated N9 is 6.7 Å from E13 and lacks neighboring proton acceptors that could allow a proton shuttle-like mechanism. Additionally, all the neutron structures presented here show E13 to be in the deprotonated, carboxylate form. Therefore, it is unlikely the observed pK_a of 5.6 is the result of E13 functioning as a general base in activating the nucleophile but instead ensures a fully deprotonated carboxylate to orient the nucleophilic water molecule properly for attack on the oxocarbenium ion intermediate. The nucleophilic water molecule then directly donates a proton to N9 subsequent to nucleophilic attack on the oxocarbenium ion intermediate. These observations of the deuterium positions of the subsequent adenine product better define the proton transfer events involved in catalysis (Fig. 3) (34).

Fig. 3.

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

The SRH-binding subsite of the HpMTAN active site contains a variety of polar residues that allow the recognition of the ribose moiety of the substrate, some of which have ambiguous hydrogen-bond donors and acceptors. In the substrate complex, the observed nucleophilic water molecule is positioned by hydrogen-bond interactions with both the guanidinium moiety of R194 and a fully deprotonated carboxylate moiety of E13 with distances of 1.8 Å and 1.6 Å, respectively (Fig. 4). The orientation of the putative nucleophile provided by these residues ensures the positioning of a lone pair of electrons in the nucleophile toward the anomeric carbon of the ribose moiety before the formation of the oxocarbenium ion intermediate. Following nucleophilic attack by the water molecule on the intermediate, a fully deprotonated E13 is observed forming a hydrogen-bond interaction with the O1′ hydroxyl of SRH in the product complex. This conserved glutamate residue has been demonstrated to be important for the catalytic reaction based on a significant loss of activity in EcMTAN variants E12Q (EcMTAN-E12Q) and E12A (6). The neutron structures described here offer an interesting alternative hypothesis for the significant, but not complete, loss of enzymatic activity in the EcMTAN variants. Inspection of each neutron structure highlights E13 in HpMTAN as a hydrogen-bond acceptor to the backbone amides of residue A9, M10, and V78 in addition to the nucleophilic water molecule. The analogous Q12 residue in EcMTAN-E12Q could alter the hydrogen-bond network for the nucleophilic water molecule and reorient it within the active site. Because of this improper orientation of the nucleophilic water molecule, the lone pair of electrons on the oxygen atom of the nucleophile needed to form the new bond with the C1′ of ribose is oriented toward the Q12 side chain in the E12Q variant and away from the oxocarbenium ion intermediate, consequently decreasing the likelihood of nucleophilic attack. This hypothesis also can explain why the EcMTAN variants retain a low level of enzymatic activity without a general base at this position. Specifically, thermal motion or random reorientation of the nucleophilic water molecule within the active site immediately following oxocarbenium ion formation in the EcMTAN-E12Q variant could allow a minority of the Michaelis complexes to undergo nucleophilic attack and complete catalysis.

Fig. 4.

Positioning of the nucleophilic water molecule in the substrate complex. The nuclear and X-ray 2Fo-Fc maps are contoured to 1σ. The nuclear 2F_O-F_C map is presented in light blue, and the X-ray 2F_O-F_C map is presented in green. The positioning of the nucleophilic water molecule is afforded by hydrogen-bonding interactions with the carboxylate moiety of E13 and the guanidinium moiety of R194. The hydrogen-bonding interactions with the two residues allow one lone pair of electrons of the water molecule to be positioned near the oxocarbenium ion intermediate to allow nucleophilic attack at the C1′ position of the ribose moiety as indicated by the arrow.

Inspection of the neutron structure for HpMTAN-D198N/SAH provides additional information that supports an expanded role of S197 in the catalytic mechanism of HpMTAN. The involvement of S197 in the catalytic reaction has been studied previously for EcMTAN with an MTAN-S197A variant that possessed 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 solvent through the classical Grotthuss mechanism (13). In the product complex, S197 and D198 develop a strong, linear, 1.9 Å, O-D⋅⋅⋅Oδ1 hydrogen-bond interaction (Fig. 5A). Although this interaction assists in positioning the D198 side chain to promote proton donation to the substrate, the presented neutron structure as well as published X-ray structures suggest that S197 may play a role in proton transfer to D198 and the substrate. In the HpMTAN-D198N/SAH neutron structure, the deuterium position on the S197 hydroxyl is reoriented, creating a new hydrogen-bond network involving a water molecule in a small hydrophobic pocket abutting the active site (Fig. 5A). The S197 O-D group rotates nearly 180°, positioning the deuterium away from the N198 side chain as the result of the δND₂ group developing an N-D⋅⋅⋅O hydrogen bond with a distance of 2.4 Å. The other δND₂ atom of N198 forms an N-D⋅⋅⋅N hydrogen bond with N7 of the SAH at a distance of 2.1 Å. In the previously published HpMTAN-D198N X-ray crystal structures, this pocket always contains a single ordered water molecule, but no corresponding density is observed in the wild-type structures (5, 12, 15). Although ordering of this water molecule could simply be a consequence of the D198N mutation, it is expected that this pocket contains a disordered water molecule in the wild-type enzyme. The water in the hydrophobic pocket interacts with S197 by accepting a hydrogen bond from the S197 side chain at a distance of 1.7 Å (Fig. 5B). Additionally, the water molecule within this small hydrophobic pocket develops a hydrogen bond with the backbone carbonyl of F208 as well as forming an O-H⋅⋅⋅π interaction with the side chain of the highly conserved F208 (35, 36). The hydrogen-bonding pattern suggests a possible mechanism for the binding of a hydronium ion within this pocket and a proton shuttle to D198 mediated by S197. Whether this water molecule plays a role in the enzyme mechanism or is simply a function of the inactivating D198N mutation is unclear. It is intriguing, however, to consider that the binding of a hydronium ion to this site in the wild-type enzyme could be stabilized through a cation-π interaction with F208 and promote D198 protonation via a proton shuttle through S197. Such a role for ordered water molecules has been highlighted in other neutron structures (37). This scenario offers a possible second mechanism by which the enzyme ensures a protonated D198 residue upon binding substrate.

Fig. 5.

The observed positions of deuterium atoms in the adenine-binding pocket of HpMTAN-D198N for the inactive binary substrate complex. In both panels, the 2F_O-F_C for the nuclear and X-ray density is contoured to 1σ and is represented in light blue and green, respectively. (A) Due to the D198N variant, the hydrogen of the hydroxyl group of S197 is reoriented to interact with a bound water molecule. (B) The interactions between the water molecule and the conserved F208 residue.

Insights into Transition-State Stabilization and Inhibitor Affinity.

In the transition-state analog neutron structures, specifically FMA and p-ClPh-Thio-DADMe-ImmA, the observed deuterium positions provide information about the specific hydrogen-bond interactions that contribute to the high-affinity binding of these inhibitors and to stabilization of the transition state. Differences in the hydrogen-bond interactions of the two transition-state analogs were observed in the ribose-binding site, whereas the hydrogen-bond interactions in the adenine-binding pocket were conserved in both transition state complexes. In the HpMTAN/FMA and HpMTAN/p-ClPh-Thio-DADMe-ImmA neutron structures, 2F_O-F_C nuclear density indicates a protonated N7 atom mimicking the protonated substrate immediately after the initiation of the catalytic reaction. The strong hydrogen-bond interaction between the deprotonated carboxylate group of D198 and the N7 proton of the nonhydrolyzable adenine mimics retains a distance of 2.0 Å and an angle of 155° between the three atoms in both complexes. Interestingly, slight variations in the hydrogen-bond distance were observed between the hydroxyl of S197 and the carboxylate of D198 in both transition-state analog structures as compared with the product complex. In the p-ClPh-Thio-DADMe-ImmA complex, the observed O-D⋅⋅⋅Oδ1 hydrogen-bond distance increased to 2.4 Å with a 142° angle. The FMA complex demonstrated a more dramatic change in the O-D⋅⋅⋅Oδ1 hydrogen bond consisting of 2.6 Å with 112° angles between the three atoms. In both the p-ClPh-Thio-DADMe-ImmA and the product complex, the hydroxyl of S197 forms a van der Waals interaction with the C8 hydrogen atom of adenine, which in FMA is replaced by a nitrogen atom. Therefore, the loss of the van der Waals interaction between the S197 hydroxyl and the substrate weakens the hydrogen bond interaction between S197 and D198.

The HpMTAN/p-ClPh-Thio-DADMe-ImmA complex demonstrates several hydrogen-bond interactions that contribute to the picomolar affinity of this inhibitor even though HpMTAN is suggested to form an early dissociative transition state. It was shown that the K_d for p-ClPh-Thio-DADMe-ImmA was 570 pM for HpMTAN, an affinity roughly 100-fold higher than that of the ImmA analog that was demonstrated to have a K_d of 40 nM (16). The differences in the affinities of the transition state analogs to structurally similar homologs of MTAN have been studied previously using computational techniques (38, 39). 2F_O-F_C nuclear density for the HpMTAN/p-ClPh-Thio-DADMe-ImmA complex demonstrates a deuterium atom located on the N1′ of the pyrrolidine moiety, confirming the presence of the cationic nitrogen atom in the catalytic site of HpMTAN (Fig. 6A). Therefore, this neutron structure gives a direct view of the hydrogen-bond interactions that orient a lone pair of electrons on the nucleophilic water molecule toward the newly formed oxocarbenium ion intermediate. The oxygen atom of the nucleophile is observed to form a strong N-D⋅⋅⋅O hydrogen-bonding interaction from both the guanidinium moiety of R194 and the cationic nitrogen atom of the N1′ from the pyrrolidine moiety with distances of 1.8 Å and 1.6 Å, respectively. Additionally, the deuterium atoms of the nucleophile form moderate-strength O-D⋅⋅⋅O hydrogen-bonding interactions with the deprotonated carboxylic acid moieties of E13 and E175 at distances of 2.3 Å and 2.5 Å, respectively. Besides the other various interactions between p-ClPh-Thio-DADMe-ImmA and HpMTAN, the observed hydrogen bonded network with the nucleophile contributes significantly to the proper orientation of the water molecule for attack on the oxocarbenium ion intermediate. To confirm that the high-affinity binding of p-ClPh-Thio-DADMe-ImmA is caused by the presence of a cationic character and not by possible flattening of the pyrrolidine ring, a 1.4-Å resolution X-ray crystal structure was refined showing that the N1′ is sp³ hybridized (Fig. S1).

Fig. 6.

The hydrogen-bonding interactions observed for the nucleophile in the presence of the fully and early dissociative transition-state analogs. The 2F_O-F_C nuclear and X-ray maps are contoured to 1σ and represented in light blue and green, respectively. (A) The deuteron positions of the nucleophile and the various hydrogen-bond interactions with p-ClPh-Thio-DADMe-ImmA are shown. Additionally, the 2F_O-F_C nuclear maps show a protonated ammonium in the pyrrolidine moiety of the DADMe-ImmA analog. (B) Positioning of the observed deuterium atoms for FMA and the unusual orientation of the nucleophile in the HpMTAN/FMA neutron structure. In the FMA complex the deuterium atom of the 3′ hydroxyl of the ribose moiety is in an unexpected position that disrupts the hydrogen-bond interaction with Oδ2 of E175 that was observed in all the other neutron structures.

Fig. S1.

The high-resolution X-ray structure of the p-ClPh-Thio-DADMe-ImmA analog demonstrates the tetrahedral geometry of the N1′ atom. The ImmA compound and active site residues coordinating the nucleophilic water molecule are shown by yellow and green carbon atoms, respectively. The F_O-F_C omit density in which the ImmA compound, each residue, and the water molecule were omitted from the map calculation is shown at 3σ.

Inspection of the HpMTAN/FMA complex provides evidence regarding the difference in affinities observed between DADMe-ImmA and ImmA transition-state analogs (Fig. 6B). In the HpMTAN/FMA complex an unusual binding orientation of the nucleophilic water molecule was observed that does not allow a lone pair of electrons to be positioned toward the inhibitor. This unusual orientation of the water molecule is afforded by strong and moderate hydrogen-bond interactions with the guanidinium moiety of R194, the carboxylate moiety of E13, and the O3′ hydroxyl of the ribose moiety of FMA. Intriguingly, in the HpMTAN/FMA neutron structure the 2F_O-F_C nuclear map shows that the deuterium atom of the O3′ hydroxyl for the ribose moiety is rotated 76° from the Oδ2 of E175 toward the 5′-alkylthio–binding subsite of the catalytic site. Although an increase in the distance between the hydrogen-bond acceptor and donor was not observed, the new proton position disrupts the hydrogen-bond interaction with Oδ2 of E175 that is observed in all other neutron structures. The new orientation of the deuterium atom for the O3′ hydroxyl allows a new O-D⋅⋅⋅O hydrogen-bond interaction to form with the O2′ hydroxyl at a distance of 2.1 Å. The O-D⋅⋅⋅Oδ2 hydrogen-bond interaction between the O3′ hydroxyl and the carboxylate side chain of E175 was shown to be essential for the catalytic reaction, because the removal of the O3′ hydroxyl from the ribose moiety and the use of E175 EcMTAN variants eliminate catalytic activity (6, 9). Based on these results, it has been proposed that the O3′ hydroxyl becomes ionized during the formation of the transition state to assist in stabilizing the oxocarbenium ion intermediate (11, 32), but the neutron structures presented here demonstrate a protonated O3′ hydroxyl and a fully deprotonated E175 carboxylate.

SI Materials and Methods

Purification of HpMTAN and HpMTAN-D198N.

Purification of both recombinant HpMTAN and HpMTAN-D198N was performed as previously described (5). Briefly, BL21 (DE3) Rosetta cells containing a pET-32–based plasmid encoding a polyhistidine–thioredoxin translational fusion with HpMTAN were grown in LB containing 0.1 mM chloramphenicol and carbenicillin at 37 °C to an absorbance at 600 nm of 0.6–0.8. Cultures were induced by the addition of 0.1 mM isopropyl β-d-1-thiogalactopyranoside and were incubated for 32 h at 16 °C. Cells were harvested by centrifugation and were resuspended in 20 mM Hepes (pH 7.5), 0.5 M sodium chloride, 5 mM β-mercaptoethanol, and 5 mM imidazole. Bacterial lysis was carried out using sonication. The lysate was centrifuged at 15,000 × g, and the filtered supernatant was applied to a 5-mL HiTrap TALON Crude column (GE Healthcare) equilibrated with buffer corresponding to the lysis buffer. A linear gradient of 5–150 mM imidazole was used to elute HpMTAN. Human rhinovirus 3C protease was added to the fractions containing HpMTAN, and the sample was dialyzed overnight against 20 mM Hepes (pH 7.5), 0.5 M sodium chloride, 5 mM β-mercaptoethanol. The protein sample then was reapplied to the 5-mL HiTrap TALON Crude column to remove the human rhinovirus 3C protease as well as the polyhistidine–thioredoxin tag. Finally, the protein sample was subjected to size-exclusion chromatography on a Hi-Load Superdex 200 column (GE Healthcare).

Crystallization Experiments.

Before crystallization, the purified HpMTAN and HpMTAN-D198N samples were dialyzed extensively against a buffer containing 20 mM Hepes (pH 7.5), 1 mM EDTA, and 0.2 mM Tris(2-carboxyethyl)phosphine (TCEP). The sample was concentrated to 50 mg/mL by ultrafiltration (EMD Millipore). The protein concentration was assessed by measuring the absorbance at 280 nm and using an extinction coefficient of 3,105 M/cm. Before crystallization, HpMTAN was incubated with SAH (Sigma), FMA (Berry and Associates, Inc.), or p-ClPh-Thio-DADMe-ImmA [synthesized by G.B.E. (40)] at a concentration of 2 mM of the compound. HpMTAN-D198N was incubated with SAH at a concentration of 2 mM to obtain the substrate complex. The crystallization studies were performed in a nine-well sandwich plate (Hampton) and equilibrated with 50 mL of well solution containing 100 mM Hepes (pH 7), 28–30% (wt/vol) PEG 550 monomethyl ether, and 50 mM magnesium chloride hexahydrate for HpMTAN complexed with SAH. The well solution for all other complexes consisted of 100 mM Hepes (pH 7.5), 15–20% (wt/vol) PEG 550 monomethyl ether, and 95 mM magnesium chloride hexahydrate. Crystallization drops consisted of 150 µL of the complex and 150 µL of the well solution. The crystals used for neutron diffraction were measured to be roughly 3.0 × 0.7 × 0.7 mm (∼1.5 mm³) and were mounted in a thick-walled quartz capillary containing a reservoir solution matching the well solution but made with 100% D₂O to allow H/D exchange before the neutron diffraction experiments.

Acknowledgments

We thank Heinz Maier-Leibnitz Zentrum and Oak Ridge National Laboratory for graciously providing the beam time that was essential for this work. This work was supported by the Center for the Advancement of Science in Space via a cooperative agreement with National Aeronautics and Space Administration Grant N-123528-01 (to D.R.R) and by National Institute of Allergy and Infectious Disease/NIH Grant AI105084 (to D.R.R.). This research used the resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and by Grant 085P1000817 from the Michigan Technology Tri-Corridor. The research was sponsored in part by the Scientific User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The IMAGINE Project was partially supported by the National Science Foundation (Grant 0922719).

Footnotes

↵¹To whom correspondence should be addressed. Email: donald.ronning{at}utoledo.edu.

Author contributions: D.R.R. designed research; M.T.B., V.M., A.O., T.E.S., and A.K. performed research; G.B.E. contributed new reagents/analytic tools; M.T.B. and D.R.R. analyzed 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 Editorial Board.
Data deposition: Crystallography, atomic coordinates, and structure factors reported in this paper have been deposited in the Protein Data Bank (PDB ID codes 5CCD, 5K1Z, 5JPC, 5CCE, and 5KB3).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1609718113/-/DCSupplemental.

View Abstract

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1. Minor W,
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3. Otwinowski Z,
4. Chruszcz M
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↵
1. Sheldrick GM
(2008) A short history of SHELX. Acta Crystallogr A 64(Pt 1):112–122.
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1. Adams PD, et al.
(2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221.
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↵
1. Adams PD,
2. Mustyakimov M,
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4. Langan P
(2009) Generalized X-ray and neutron crystallographic analysis: More accurate and complete structures for biological macromolecules. Acta Crystallogr D Biol Crystallogr 65(Pt 6):567–573.
.
OpenUrl CrossRef PubMed
↵
1. Emsley P,
2. Lohkamp B,
3. Scott WG,
4. Cowtan K
(2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501.
.
OpenUrl CrossRef PubMed
↵
1. Gerlits O, et al.
(2016) Long-range electrostatics-induced two-proton transfer captured by neutron crystallography in an enzyme catalytic site. Angewandte Chemie 55:4924–4927.
.
OpenUrl
↵
1. Singh V,
2. Lee JE,
3. Núñez S,
4. Howell PL,
5. Schramm VL
(2005) Transition state structure of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues. Biochemistry 44(35):11647–11659.
.
OpenUrl CrossRef PubMed
↵
1. Lee JE,
2. Cornell KA,
3. Riscoe MK,
4. Howell PL
(2003) Structure of Escherichia coli 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase inhibitor complexes provide insight into the conformational changes required for substrate binding and catalysis. J Biol Chem 278(10):8761–8770.
.
OpenUrl Abstract/FREE Full Text
↵
1. Ronning DR,
2. Iacopelli NM,
3. Mishra V
(2010) Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase. Protein Sci 19(12):2498–2510.
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(2011) Direct measurements of electric fields in weak OH···π hydrogen bonds. J Am Chem Soc 133(43):17414–17419.
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(1999) The Weak Hydrogen Bond (Oxford Univ Press, New York).
.
↵
1. Kovalevsky AY, et al.
(2011) Identification of the elusive hydronium ion exchanging roles with a proton in an enzyme at lower pH values. Angew Chem Int Ed Engl 50(33):7520–7523.
.
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↵
1. Zoi I,
2. Motley MW,
3. Antoniou D,
4. Schramm VL,
5. Schwartz SD
(2015) Enzyme homologues have distinct reaction paths through their transition states. J Phys Chem B 119(9):3662–3668.
.
OpenUrl
↵
1. Motley MW,
2. Schramm VL,
3. Schwartz SD
(2013) Conformational freedom in tight binding enzymatic transition-state analogues. J Phys Chem B 117(33):9591–9597.
.
OpenUrl
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1. Evans GB, et al.
(2005) Second generation transition state analogue inhibitors of human 5′-methylthioadenosine phosphorylase. J Med Chem 48(14):4679–4689.
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Proceedings of the National Academy of Sciences: 113 (48)

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Article Classifications

Biological Sciences
Biochemistry

[1] ↵
Dairi T
(2009) An alternative menaquinone biosynthetic pathway operating in microorganisms: An attractive target for drug discovery to pathogenic Helicobacter and Chlamydia strains. J Antibiot (Tokyo) 62(7):347–352.
.
OpenUrl CrossRef PubMed

[2] Dairi T

[3] ↵
Thomas K, et al.
(2012) Femtomolar inhibitors bind to 5′-methylthioadenosine nucleosidases with favorable enthalpy and entropy. Biochemistry 51(38):7541–7550.
.
OpenUrl

[4] Thomas K, et al.

[5] ↵
Wang S, et al.
(2012) A picomolar transition state analogue inhibitor of MTAN as a specific antibiotic for Helicobacter pylori. Biochemistry 51(35):6892–6894.
.
OpenUrl CrossRef PubMed

[6] Wang S, et al.

[7] ↵
Wang S, et al.
(2015) New antibiotic candidates against Helicobacter pylori. J Am Chem Soc 137(45):14275–14280.
.
OpenUrl

[8] Wang S, et al.

[9] ↵
Mishra V,
Ronning DR
(2012) Crystal structures of the Helicobacter pylori MTAN enzyme reveal specific interactions between S-adenosylhomocysteine and the 5′-alkylthio binding subsite. Biochemistry 51(48):9763–9772.
.
OpenUrl CrossRef PubMed

[10] Mishra V,

[11] Ronning DR

[12] ↵
Lee JE, et al.
(2005) Mutational analysis of a nucleosidase involved in quorum-sensing autoinducer-2 biosynthesis. Biochemistry 44(33):11049–11057.
.
OpenUrl CrossRef PubMed

[13] Lee JE, et al.

[14] ↵
Parveen N,
Cornell KA
(2011) Methylthioadenosine/S-adenosylhomocysteine nucleosidase, a critical enzyme for bacterial metabolism. Mol Microbiol 79(1):7–20.
.
OpenUrl CrossRef PubMed

[15] Parveen N,

[16] Cornell KA

[17] ↵
Della Ragione F,
Porcelli M,
Cartenì-Farina M,
Zappia V,
Pegg AE
(1985) Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action. Biochem J 232(2):335–341.
.
OpenUrl Abstract/FREE Full Text

[18] Della Ragione F,

[19] Porcelli M,

[20] Cartenì-Farina M,

[21] Zappia V,

[22] Pegg AE

[23] ↵
Allart B,
Gatel M,
Guillerm D,
Guillerm G
(1998) The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli-chemical evidence for a transition state with a substantial oxocarbenium character. Eur J Biochem 256(1):155–162.
.
OpenUrl PubMed

[24] Allart B,

[25] Gatel M,

[26] Guillerm D,

[27] Guillerm G

[28] ↵
Lee JE, et al.
(2005) Structural rationale for the affinity of pico- and femtomolar ransition state analogues of Escherichia coli 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase. J Biol Chem 280(18):18274–18282.
.
OpenUrl Abstract/FREE Full Text

[29] Lee JE, et al.

[30] ↵
Singh V,
Schramm VL
(2007) Transition-state analysis of S. penumoniae 5′-methylthioadenosine nucleosidase. J Am Chem Soc 129(10):2783–95.
.
OpenUrl CrossRef PubMed

[31] Singh V,

[32] Schramm VL

[33] ↵
Lee JE, et al.
(2005) Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis. J Mol Biol 352(3):559–574.
.
OpenUrl CrossRef PubMed

[34] Lee JE, et al.

[35] ↵
Lee JE,
Cornell KA,
Riscoe MK,
Howell PL
(2001) Structure of E. coli 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases. Structure 9(10):941–953.
.
OpenUrl CrossRef PubMed

[36] Lee JE,

[37] Cornell KA,

[38] Riscoe MK,

[39] Howell PL

[40] ↵
Harris T,
Turner GJ
(2002) Structural basis of perturbed pKa values of catalytic groups in enzyme active sites. IUBMB Life 53(2):85–98.
.
OpenUrl CrossRef PubMed

[41] Harris T,

[42] Turner GJ

[43] ↵
Kim RQ,
Offen WA,
Davies GJ,
Stubbs KA
(2014) Structural enzymology of Helicobacter pylori methylthioadenosine nucleosidase in the futalosine pathway. Acta Crystallogr D Biol Crystallogr 70(Pt 1):177–185.
.
OpenUrl CrossRef

[44] Kim RQ,

[45] Offen WA,

[46] Davies GJ,

[47] Stubbs KA

[48] ↵
Gutierrez JA, et al.
(2007) Picomolar inhibitors as transition-state probes of 5′-methylthioadenosine nucleosidases. ACS Chem Biol 2(11):725–734.
.
OpenUrl CrossRef PubMed

[49] Gutierrez JA, et al.

[50] ↵
Singh V, et al.
(2005) Femtomolar transition state analogue inhibitors of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli. J Biol Chem 280(18):18265–18273.
.
OpenUrl Abstract/FREE Full Text

[51] Singh V, et al.

[52] ↵
Myles DA
(2006) Neutron protein crystallography: Current status and a brighter future. Curr Opin Struct Biol 16(5):630–637.
.
OpenUrl CrossRef PubMed

[53] Myles DA

[54] ↵
Ehrlich JI,
Schramm VL
(1994) Electrostatic potential surface analysis of the transition state for AMP nucleosidase and for formycin 5′-phosphate, a transition-state inhibitor. Biochemistry 33(30):8890–8896.
.
OpenUrl

[55] Ehrlich JI,

[56] Schramm VL

[57] ↵
Otwinowski Z, et al.
(1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326.
.
OpenUrl CrossRef PubMed

[58] Otwinowski Z, et al.

[59] ↵
Meilleur F, et al.
(2013) The IMAGINE instrument: First neutron protein structure and new capabilities for neutron macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 69(Pt 10):2157–2160.
.
OpenUrl CrossRef

[60] Meilleur F, et al.

[61] ↵
Campbell JW
(1995) LAUEGEN, an X-windows-based program for the processing of Laue diffraction data. J Appl Cryst 28:228–236.
.
OpenUrl CrossRef

[62] Campbell JW

[63] ↵
Campbell JWH,
Hao Q,
Harding MM,
Nguti ND,
Wilkinson C
(1998) LAUEGEN version 6.0 and INTLDM. J Appl Cryst 31:496–502.
.
OpenUrl CrossRef

[64] Campbell JWH,

[65] Hao Q,

[66] Harding MM,

[67] Nguti ND,

[68] Wilkinson C

[69] ↵
Arzt SC,
Campbell JW,
Harding MM,
Hao Q,
Helliwell JR
(1999) LSCALE - the new normalization, scaling and absorption correction program in the Daresbury Laue software suit. J Appl Cryst 32:554–562.
.
OpenUrl CrossRef

[70] Arzt SC,

[71] Campbell JW,

[72] Harding MM,

[73] Hao Q,

[74] Helliwell JR

[75] ↵
Weiss MS
(2001) Global indicators of X-ray data quality. J Appl Cryst 34:130–135.
.
OpenUrl CrossRef

[76] Weiss MS

[77] ↵
Minor W,
Cymborowski M,
Otwinowski Z,
Chruszcz M
(2006) HKL-3000: The integration of data reduction and structure solution--from diffraction images to an initial model in minutes. Acta Crystallogr D Biol Crystallogr 62(Pt 8):859–866.
.
OpenUrl CrossRef PubMed

[78] Minor W,

[79] Cymborowski M,

[80] Otwinowski Z,

[81] Chruszcz M

[82] ↵
Sheldrick GM
(2008) A short history of SHELX. Acta Crystallogr A 64(Pt 1):112–122.
.
OpenUrl CrossRef PubMed

[83] Sheldrick GM

[84] ↵
Adams PD, et al.
(2010) PHENIX: A comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66(Pt 2):213–221.
.
OpenUrl CrossRef PubMed

[85] Adams PD, et al.

[86] ↵
Adams PD,
Mustyakimov M,
Afonine PV,
Langan P
(2009) Generalized X-ray and neutron crystallographic analysis: More accurate and complete structures for biological macromolecules. Acta Crystallogr D Biol Crystallogr 65(Pt 6):567–573.
.
OpenUrl CrossRef PubMed

[87] Adams PD,

[88] Mustyakimov M,

[89] Afonine PV,

[90] Langan P

[91] ↵
Emsley P,
Lohkamp B,
Scott WG,
Cowtan K
(2010) Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66(Pt 4):486–501.
.
OpenUrl CrossRef PubMed

[92] Emsley P,

[93] Lohkamp B,

[94] Scott WG,

[95] Cowtan K

[96] ↵
Gerlits O, et al.
(2016) Long-range electrostatics-induced two-proton transfer captured by neutron crystallography in an enzyme catalytic site. Angewandte Chemie 55:4924–4927.
.
OpenUrl

[97] Gerlits O, et al.

[98] ↵
Singh V,
Lee JE,
Núñez S,
Howell PL,
Schramm VL
(2005) Transition state structure of 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase from Escherichia coli and its similarity to transition state analogues. Biochemistry 44(35):11647–11659.
.
OpenUrl CrossRef PubMed

[99] Singh V,

[100] Lee JE,

[101] Núñez S,

[102] Howell PL,

[103] Schramm VL

[104] ↵
Lee JE,
Cornell KA,
Riscoe MK,
Howell PL
(2003) Structure of Escherichia coli 5′-methylthioadenosine/ S-adenosylhomocysteine nucleosidase inhibitor complexes provide insight into the conformational changes required for substrate binding and catalysis. J Biol Chem 278(10):8761–8770.
.
OpenUrl Abstract/FREE Full Text

[105] Lee JE,

[106] Cornell KA,

[107] Riscoe MK,

[108] Howell PL

[109] ↵
Ronning DR,
Iacopelli NM,
Mishra V
(2010) Enzyme-ligand interactions that drive active site rearrangements in the Helicobacter pylori 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase. Protein Sci 19(12):2498–2510.
.
OpenUrl CrossRef PubMed

[110] Ronning DR,

[111] Iacopelli NM,

[112] Mishra V

[113] ↵
Saggu M,
Levinson NM,
Boxer SG
(2011) Direct measurements of electric fields in weak OH···π hydrogen bonds. J Am Chem Soc 133(43):17414–17419.
.
OpenUrl CrossRef PubMed

[114] Saggu M,

[115] Levinson NM,

[116] Boxer SG

[117] ↵
Desiraju GST
(1999) The Weak Hydrogen Bond (Oxford Univ Press, New York).
.

[118] Desiraju GST

[119] ↵
Kovalevsky AY, et al.
(2011) Identification of the elusive hydronium ion exchanging roles with a proton in an enzyme at lower pH values. Angew Chem Int Ed Engl 50(33):7520–7523.
.
OpenUrl CrossRef PubMed

[120] Kovalevsky AY, et al.

[121] ↵
Zoi I,
Motley MW,
Antoniou D,
Schramm VL,
Schwartz SD
(2015) Enzyme homologues have distinct reaction paths through their transition states. J Phys Chem B 119(9):3662–3668.
.
OpenUrl

[122] Zoi I,

[123] Motley MW,

[124] Antoniou D,

[125] Schramm VL,

[126] Schwartz SD

[127] ↵
Motley MW,
Schramm VL,
Schwartz SD
(2013) Conformational freedom in tight binding enzymatic transition-state analogues. J Phys Chem B 117(33):9591–9597.
.
OpenUrl

[128] Motley MW,

[129] Schramm VL,

[130] Schwartz SD

[131] ↵
Evans GB, et al.
(2005) Second generation transition state analogue inhibitors of human 5′-methylthioadenosine phosphorylase. J Med Chem 48(14):4679–4689.
.
OpenUrl CrossRef PubMed

[132] Evans GB, et al.

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Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Significance

Abstract

Materials and Methods

Neutron Crystallography.

X-Ray Crystallography.

Joint XN Refinement.

Results and Discussion

Defining Proton Positions During the Catalytic Reaction of HpMTAN.

Insights into Transition-State Stabilization and Inhibitor Affinity.

SI Materials and Methods

Purification of HpMTAN and HpMTAN-D198N.

Crystallization Experiments.

Acknowledgments

Footnotes

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Neutron structures of the Helicobacter pylori 5′-methylthioadenosine nucleosidase highlight proton sharing and protonation states

Significance

Abstract

Materials and Methods

Neutron Crystallography.

X-Ray Crystallography.

Joint XN Refinement.

Results and Discussion

Defining Proton Positions During the Catalytic Reaction of HpMTAN.

Insights into Transition-State Stabilization and Inhibitor Affinity.

SI Materials and Methods

Purification of HpMTAN and HpMTAN-D198N.

Crystallization Experiments.

Acknowledgments

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