Mycobacterial OtsA Structures Unveil Substrate Preference Mechanism and Allosteric Regulation by 2-Oxoglutarate and 2-Phosphoglycerate

Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria.

In mycobacteria, trehalose is also an essential component of mycolic acids and other cell wall glycolipids, which are major protagonists in pathogenesis of Mycobacterium tuberculosis (1), the causative agent of the widespread infectious disease tuberculosis. In these organisms, trehalose was further identified as a key signaling molecule of cell envelope stress, playing a role as an activator of the iniBAC operon, which is induced when mycobacteria are exposed to the first-line drug isoniazid (21).
All mycobacterial species, with a few exceptions, possess two pathways to synthesize trehalose, the OtsAB and the TreYZ pathways (1). The OtsAB pathway is the most widely distributed trehalose biosynthesis pathway, present in bacteria, archaea, and eukaryotes (22,23). In this pathway, which is conserved and essential in mycobacteria, trehalose is synthesized in a two-step process involving OtsA and OtsB2 enzymes. M. tuberculosis mutants showed that the OtsAB pathway was not only the dominant route for trehalose biosynthesis in this pathogen but also required for growth both in vitro and in a mouse infection model, with marked growth and virulence defects of OtsA mutants (24) and strict essentiality of OtsB2 due to the toxic effect of trehalose-6phosphate (T6P) accumulation (24,25).
OtsA, a glycosyltransferase that belongs to the GT20 family of the CAZY classification (www.cazy.org), uses the ␣ anomer of glucose-6-phosphate (G6P) as acceptor and nucleoside diphosphate glucose (NDP-glucose) as the donor to synthesize T6P, with net retention of the anomeric configuration of the donor substrate (26,27). Binding of the substrates occurs sequentially in a "bi-bi mechanism" with NDP-glucose binding first and G6P second (26,28). Interestingly, OtsAs from different organisms show different donor preferences as shown by kinetic studies and X-ray crystal structures, but the reasons behind these preferences are poorly understood (27,(29)(30)(31)(32)(33). Recently, different roles were identified for OtsA beyond its enzymatic activity. OtsA was reported to act as an osmotic stress sensor and morphogenetic protein that can regulate the switch to mycelioid growth in Arthrobacter sp. strain A3, a pleomorphic soil-dwelling actinobacterium (34).
In this work, we purified, crystallized, and solved the structure of Mycobacterium thermoresistibile OtsA (MtrOtsA). To gain further insight into the substrate preference of mycobacterial OtsAs, we have obtained structures with substrates that define the mechanisms of ADP-glucose preference. Structure-guided point mutations of key residues in the active site were performed, and characterization of the mutants showed that three mutations are enough to change the donor substrate preference to UDPglucose. Further structures with OtsA product and pathway product were obtained, showing how this enzyme is feedback inhibited by trehalose. Importantly, with these structures, we have also identified a new allosteric site and allosteric regulators of this enzyme that link glycolytic and tricarboxylic acid (TCA) cycle metabolites to the regulation of trehalose synthesis.
ing that this domain is more dynamic, which is consistent with the large movements of ␣-1 during catalytic activity.
MtrOtsA forms a tetramer in solution, as previously observed for M. tuberculosis OtsA. This tetrameric form is also observed in all the crystal structures reported here (Fig. 1B). The amino acids involved in the tetramer interfaces are not conserved beyond mycobacteria and related species (Fig. S2), suggesting that OtsA might have a different molecular assembly in different species. Indeed, in E. coli both tetrameric and dimeric forms were reported (27) while in Streptomyces venezuelae and Aspergillus fumigatus only the dimeric form was observed (29,30). However, in mycobacteria and closely related organisms the tetramer interfaces are highly conserved, suggesting a tetrameric assembly of OtsA in all of these organisms (Fig. S2).
Catalytic site. OtsA is a glycosyltransferase that uses the ␣ anomer of G6P as acceptor and NDP-glucose as the donor to synthesize T6P. The MtrOtsA catalytic site is located between the two Rossmann fold domains in a large and deep cavity. Structures with donor substrates were determined by soaking the apo form crystals with ADPglucose and GDP-glucose (Fig. 2). The donor substrates interact primarily with the C-terminal domain through the side chains of the highly conserved Arg286, Lys291, Asp385, and Glu393 and with the side chain of Arg365 (Fig. 2), which is conserved in all mycobacteria and closely related species but less conserved outside this group (Fig. S2). Backbone interactions with the absolutely conserved residue Gly386, Met387, Asn388, and Leu389 amine groups are also observed. Similar interactions were observed before for E. coli OtsA (27,32). While the adenine moiety interacts with the Val363 amino group, the Leu319 carbonyl, and a coordinated water ( Fig. 2A), the guanine moiety forms interactions with Val363 only (Fig. 2B). N-terminal domain interactions with the donor substrate are observed only for His168 when the active site is in an open conformation (Fig. 2). To obtain a structure with the acceptor substrate, we cocrystallized OtsA in the presence of 5 mM ADP and G6P. As G6P binds to the protein in the presence of a donor substrate or its nucleotide, the active site adopts a closed conformation (Fig. 3A). The OtsA:ADP:G6P ternary complex crystallized in the P6 2 22 space group, with one protomer per asymmetric unit, and the crystals diffracted up to ϳ1.7-Å resolution. In this closed conformation, new interactions with the donor substrate are formed with the Gly39 peptide-NH function (Fig. 3). Due to the absolute conserved nature of Gly39 among all functional OtsAs and related enzymes (27) and the contact that it forms with the glucose-bound phosphate, this residue is highly likely to be mechanistically involved in the catalytic activity (27).
The acceptor interactions recapitulate what was observed before for E. coli (27) with the substrate interacting with the highly conserved residues Arg18, Tyr90, Asp144, and Gln146 of the N-terminal domain and with only a single residue of the C-terminal domain, Arg324 (Fig. 3B). The residues involved in acceptor substrate interaction are highly conserved across both bacteria and yeast (Fig. S2). As described for E. coli OtsA (27), the OtsA:ADP:G6P ternary structure shows the catalytic site in a closed conformation that substantially differs from the apo form, with ␣-helix 1 and the Arg35-Gly39 loop region moving up to ϳ12 Å (Fig. 3A).
Properties of M. thermoresistibile OtsA. MtrOtsA uses ADP-glucose, UDP-glucose, and GDP-glucose as glucose donors with a preference for ADP-glucose (Table 1) and G6P as the only acceptor, in accordance with what was reported before for M. tuberculosis OtsA (33,35). The kinetic parameters for MtrOtsA were obtained and are reported in Table 1 and Fig. S3 with K m app values for the preferred donor substrate (ADP-glucose) of 0.25 Ϯ 0.02 mM and for the acceptor (G6P) of 3.3 Ϯ 0.1 mM. The K m app for GDP-glucose of 0.29 Ϯ 0.02 mM was in the same range as the one obtained for ADP-glucose; however, the turnover was 5-fold lower ( Table 1). For UDP-glucose, the enzyme showed an ϳ7-fold-higher K m app of 1.7 Ϯ 0.1 mM than the one obtained for ADP-glucose ( Table 1).
The preference of ADP-glucose over the other glucose donors was further confirmed by isothermal titration calorimetry (ITC). The binding affinity of substrate donors could be determined only for ADP-glucose under the tested conditions with an observable K d (dissociation constant) of 27.17 Ϯ 2.66 M (Fig. S4). Other glucose donors analyzed (GDP-glucose and UDP-glucose) had no observable heat of binding, and the same was observed for G6P. The lack of heat of binding for GDP-and UDP-glucose is most likely due to their reduced affinity. In the case of G6P, it reflects a necessity of previous binding of the donor substrate.
Donor substrate preference is largely mediated by three residues. OtsAs from different organisms have been shown to have different donor substrate preferences, while being capable of using several nucleotide donors (30,33,36,37), which is reflected in lower conservation of the donor substrate-interacting residues (Fig. S2). Preference for ADP-glucose as the donor substrate in MtrOtsA is conferred by interactions with the deeply buried adenine moiety (Fig. 4A). The carbonyl groups of Leu319 and Arg361 interact with the primary amine, and the amide group of Val363 interacts with N-1 of the adenine moiety (Fig. 4A). A highly coordinated water interacts with the primary amine of the adenine moiety and the carbonyl groups of Leu359 and Arg361. The guanine moiety of GDP-glucose does not occupy the same deeply buried pocket as the adenine because its primary amine group would sterically clash with the Val363 carbonyl group (Fig. 4B). It thus binds to OtsA more weakly than ADP-glucose, explaining the substrate preference and consequently the lack of observable heat of binding under the tested ITC conditions. A binary complex structure with OtsA and UDPglucose was also obtained, but the electron density was observed for only glucose and the two phosphates (not shown), indicating a reduced preference for this substrate as confirmed by enzymatic and biophysical data. Comparing the OtsA structures of M. thermoresistibile and E. coli, we hypothesized that the ADP preference was mediated by the substitution of an isoleucine (Ile295 in E. coli) for a leucine (Leu319 in M. thermoresistibile). The presence of the leucine in M. thermoresistibile allows the primary amine of the adenine moiety to occupy a buried position, interacting with the carbonyl group of Leu319 ( Fig. 4C) which no other nucleotide activated donor can occupy. However, given the differences between the two enzymes, it is likely that other residues could also play a role. To improve the selection of residues to mutate, we employed a computational approach using mCSM-lig (38), a software program developed by our group that predicts the effects of mutations on binding affinity. The software predicted that several residues within 4.5 Å of the adenine moiety have a destabilizing effect on the interactions between M. thermoresistibile OtsA and ADP-glucose if mutated to the E. coli OtsA equivalent (Table S2). Combining this information with structural analysis, three mutations predicted to be destabilizing were selected. These mutations are Val363Phe, Leu319Ile, and Glu367Leu, the last being a long-distance mutation that we hypothesized would have a strong effect on ligand binding.
To confirm our hypothesis, we generated several MtrOtsA mutants (Leu319Ile, Val363Phe, Leu319Ile-Glu367Leu, and a triple mutant) and obtained kinetic data for these mutants ( Table 1). As predicted, the mutant Leu319Ile showed an ϳ3-fold increase in the K m app for ADP-glucose whereas K m app values were similar for GDPglucose and UDP-glucose compared to the wild type. Even though GDP-glucose now showed the best K m app among the three different donors, the catalytic efficiency and turnover rate were still higher for ADP-glucose (Table 1). A complete reversal of the donor substrate preference for UDP-glucose was obtained only with a combination of the three mutations ( Table 1). The triple mutant showed an ϳ5-fold increase in ADP-glucose K m app and a reduction for the UDP-glucose K m app by more than 3-fold ( Table 1). The catalytic efficiency was also completely reversed for ADP-and UDPglucose between the wild type and the triple mutant (Table 1). Although Leu319Ile showed the largest contribution to ADP-glucose K m app increase, Val363Phe and Glu367Leu were also determinant for the reversal to UDP-glucose preference (Table 1). This can be explained by the fact that the phenylalanine substitution at position 363 forces the loop between ␤-14 and ␣-14 to move toward the nucleotide binding site (Fig. 4C), thus establishing stronger interactions with a pyrimidine nucleotide and clashing with purine nucleotides. This effect is also observed for both ADP-and GDP-glucose, with an ϳ2.5-fold increase in K m app over the wild type. The leucine substitution at position 367 further helps in this move since the hydrogen bonds between the side chains of Arg266 and Glu367 are no longer present and ␣-14 is repelled from Arg266 due to the Leu side chain (Fig. 4C).
Feedback inhibition. We soaked both trehalose and T6P into apo OtsA crystals and solved structures with both ligands (Fig. 4D and E). In both structures, the OtsA active site assumes an open conformation and two compounds superpose almost perfectly at the active site, recapitulating all of the interactions for the donor glucose and also interacting with His199 and Arg286, the latter a residue that interacts with the phosphates of the donor substrate ( Fig. 4D and E). The structure with T6P further shows the phosphate group of the product occupying a similar position as the phosphate group of G6P, interacting with the side chains of Tyr90, Gln146, Arg324, and Tyr145, a residue that does not interact with G6P (Fig. 4E). Interestingly, the buffer present under the crystallization condition, N-cyclohexyl-2-aminoethanesulfonic acid (CHES), is observed in both trehalose-and trehalose-6-phosphate-bound structures, occupying the site of the nucleotide donor with the sulfate group binding in a phosphate site and interacting with hydroxyl groups from both glucose units of trehalose (Fig. 4D and E). After obtaining these structures, we expected to observe some degree of feedback inhibition of OtsA by trehalose given that, under physiological conditions, free trehalose is highly abundant inside mycobacterial cells, reaching up to 15% of the total organic material in actively growing Mycobacterium smegmatis cells (39), while T6P could not be detected in M. tuberculosis cell extracts (25). Trehalose was found to inhibit the enzyme with a 50% inhibitory concentration (IC 50 ) of ϳ24 mM (Fig. S5). These results suggest that under physiological conditions, OtsA can be regulated by trehalose, the final product of the pathway which is highly abundant inside mycobacterial cells (39).
Identification of an allosteric site in M. thermoresistibile OtsA. Trehalose is a multipurpose molecule that is abundant in mycobacteria. It not only can function as an energy reserve and as structural component of cell wall glycolipids but can also lead to the synthesis of glycogen through the TreS-Mak-GlgE pathway and be directly synthesized from glycogen from the TreX-TreY-TreZ pathway. It is therefore likely that trehalose synthesis through the OtsA-B pathway is under strong regulation.

Mycobacterial OtsA Substrate Preference and Regulation
® Under all the crystallization conditions that contained CHES, we could observe this compound in the crystal structures occupying a pocket formed by the contact interface of two OtsA protomers in the tetrameric assembly (Fig. 5). The CHES sulfate group directly interacts with the side chains of Arg384 of protomer A and Arg213 of protomer B, forming hydrogen bonds with both but also hydrogeninteractions with Phe410 of protomer A (Fig. 5). The residues composing this site are completely conserved in mycobacterial and closely related species that are likely to harbor OtsA tetramers but not in other species known to have other oligomeric forms (Fig. S2).
Arg384 sits in a loop that forms extensive contacts with the donor substrate through Asp385, Gly386, Met387, and Leu389 (Fig. 2). Ligands interacting with the side chain of Arg384 could therefore have an impact on the activity of the enzyme, making this site a prime candidate for allosteric regulation.
While CHES had no discernible impact on OtsA activity at the tested concentrations, it was reported before that F6P acted as an allosteric regulator of M. tuberculosis OtsA (35), and the same was reported for the yeast OtsA-B complex (40). We have tested MtrOtsA under the same conditions as those reported by Asencion Diez and colleagues (35) and could not detect any effect of F6P for MtrOtsA. Although we did not observe any effect of F6P on enzyme activity for MtrOtsAs, we nevertheless attempted to soak this compound in both apo form and OtsA:ADP:G6P ternary complex crystals and also cocrystallize it in the presence and absence of ADP and G6P, all under CHES-free conditions. None of these conditions provided a structure in which F6P occupied an allosteric site. We could, however, observe F6P when cocrystallized together with ADP, bound to the acceptor substrate site, with OtsA presenting the active site in a closed conformation (Fig. S6).
From the several compounds tested, only 2OG and 2PG showed clear inhibition of OtsA activity with IC 50 s of 1.8 mM and 2.3 mM, respectively ( Table 2 and Fig. 5C). To assess whether they could be acting allosterically on MtrOtsA and binding to the site identified by CHES, we performed two mutations on the arginines that interacted with CHES (Arg213Glu and Arg384Glu) and tested the two mutants' activity in the presence of 2OG and 2PG. The results show that the Arg213Glu mutation abolished the strong response of the protein to 2OG while there was an ϳ5-fold increase in 2PG IC 50 to 12 mM. For the Arg384Glu mutant, 2OG unexpectedly becomes a strong activator with a 50% effective concentration (EC 50 ) of 0.7 mM while 2PG has no effect ( Table 2).

DISCUSSION
Trehalose, an essential disaccharide in mycobacteria, is one of the critical components of the cell wall. The OtsA-B pathway is an essential source of trehalose for these organisms, with knockouts of this pathway being growth defective or nonviable (24,25). Here, we reveal the first mycobacterial OtsA structure. The overall structure of M. thermoresistibile OtsA is similar to the reported structures of E. coli, S. venezuelae, and Paraburkholderia xenovorans OtsAs despite relatively low sequence identity (30 to 35%). Remarkably, OtsAs also have a fold highly similar to the pseudoglycosyltransferases VldE and ValL involved in synthesis of validamycin A, a potent antifungal agent (43,44).
Nevertheless, there are substantial differences in the oligomeric organization of bacterial OtsAs, with tetrameric, dimeric, and protomeric forms reported. While mycobacteria and related species present conservation of interfaces, showing that the observed tetrameric form for MtrOtsA is present in all these organisms, beyond this group such conservation is absent, which is consistent with other oligomeric states (see Fig. S2 in the supplemental material).
The structures obtained in this work allow us to explain how mycobacterial OtsA prefers ADP-glucose as a substrate. A cavity found to accommodate the binding of the adenine moiety is surrounded with Leu319 and Arg361, which are critical for substrate recognition. It is further mediated by the Val363 carbonyl group, which does not allow GDP-glucose to occupy the same position, and by Glu367. Mutating these three residues to the E. coli OtsA equivalents completely changed the preference to UDPglucose, the preferred E. coli OtsA donor substrate. Both Leu319 and Glu367 are highly conserved in all mycobacterial and closely related species analyzed but not beyond this group, while Val363 can be replaced by other small hydrophobic amino acids, such as alanine and isoleucine, in other mycobacterial species and closely related species (Fig. S2). However, outside this group Val363 is replaced by bulkier residues, such as tyrosine and phenylalanine (Fig. S2). These differences are suggestive of alternative donor substrate preferences for organisms outside the suborder Corynebacterineae.
Trehalose is not only abundant inside the mycobacterial cell but also involved in a cycle that links it to the synthesis and degradation of glycogen (45). We have shown that OtsA is feedback inhibited by trehalose but not by T6P, even though the structures obtained show that both compounds form extensive interactions with OtsA. However, given that these two structures were obtained by soaking apo-form crystals with high concentrations of trehalose and T6P and that CHES is also observed at the active site interacting with both compounds, it is possible that these two structures do not entirely reflect their natural interactions with the enzyme. The toxic accumulation of T6P in OtsB2-knockout mutants (25) can be explained by these results, since OtsA shows very low sensitivity to T6P. Furthermore, trehalose levels are reduced in the mutant (25), further contributing to an increase in OtsA activity, leading to higher T6P production. Regulation of OtsA by phosphorylation and methylation has been observed for yeast trehalose-6-phosphate synthase complex (46,47), and phosphorylation of E. coli OtsA has also been reported (48) in two residues close to the active site. Even though only one of the residues is conserved in mycobacteria (Ser323 for MtrOtsA), phosphorylation of this residue would most likely lead to inactivation of the enzyme due to the location close to the acceptor binding site (Fig. S7). Interestingly, this residue is highly conserved in all sequences analyzed, with a single exception where it is replaced by a threonine (Fig. S2), suggesting that phosphorylation of this residue might be a common regulatory mechanism of OtsA activity.
Allosteric regulation was reported for both M. tuberculosis OtsA and yeast trehalose phosphate synthase complex, mediated by F6P (35,40). The structure obtained with F6P shows this compound occupying the donor site, in the presence of ADP, with the enzyme adopting a closed conformation. Nevertheless, OtsA could use only G6P as an acceptor, and F6P showed no effect on the enzyme activity, indicating that the presence of F6P in the acceptor site was forced by the high concentration used for cocrystallization (5 mM). The observed differences in regulation by F6P between mycobacterial enzymes are difficult to reconcile since the allosteric site is highly conserved in mycobacterial OtsAs and closely related species (Fig. S2).
MtrOtsA is allosterically regulated by 2PG, the substrate of enolase, a glycolytic enzyme, and 2OG, a master metabolic regulator that sits at the interface of carbon and nitrogen metabolism. The observed effect is within relevant physiological concentrations for both compounds (49,50).
The effect of 2PG on OtsA and the reported interaction with enolase suggest an interplay between these two enzymes that would be interesting to explore further on the enolase side. Enolase is a multifunctional protein involved in a variety of cellular processes, beyond its enzymatic activity, which include response to oxidative and thermal stress (51). Given that trehalose is a known chemical chaperone and compatible solute (1), the association between enolase and OtsA, through the effect of 2PG on OtsA activity and the reported interaction between the two enzymes, points to further regulation of stress response.
2OG is a molecule that sits at the interface of carbon and nitrogen metabolism and that has been shown to regulate many different pathways (42). The role of trehalose as an energy reserve molecule (1) and its relationship with synthesis and degradation of glycogen (52) point to a mechanism in which 2OG influences the synthesis of these two compounds through regulation of OtsA activity, one that deserves to be further explored.
We have shown that OtsA can be allosterically inhibited; however, a single mutation at the allosteric site changed the behavior of 2OG, but not 2PG, from an inhibitor to an activator. This hints at the possible existence of allosteric activators of OtsA yet to be discovered.
The conservation of allosteric site residues and oligomeric assembly in the suborder Corynebacterineae but not outside suggests that allosteric regulation of OtsA through this site might be limited to this group of organisms. The results of this work are a significant step forward in understanding the regulation of trehalose synthesis in mycobacteria and the structural reasons behind substrate preference and provide new important insight into this enzyme.
To assess the specificity of OtsA for G6P, several compounds (F6P, mannose-6-phosphate, glucosamine-6-phosphate, and G1P) were tested for their capacity to act as acceptor substrates, under the conditions above, using ADP-glucose as the donor substrate. The effect of trehalose and T6P as feedback inhibitors and of G1P, F6P, F16BP, CHES, 2PG, 3PG, cAMP, and 2OG as allosteric regulators was tested under the conditions described above but with fixed concentrations of G6P and ADP-glucose (both 0.3 mM). The same conditions were used to test possible allosteric regulators for wild-type OtsA and mutants Arg213Glu and Arg384Glu. All experiments were performed in triplicate in a PHERAstar plate reader (BMG Labtech), and data were analyzed with Prism 5 (GraphPad Software).
To assess the inhibitory effect of ADP and the allosteric effect of phosphoenolpyruvate, an endpoint assay was performed instead. Reaction mixtures (100 l) containing 50 mM Tris (pH 7.5), 200 mM NaCl, 10 mM MgCl 2 , 50 mM KCl, 4 M MtrOtsA, 0.3 mM ADP-glucose, 0.3 mM G6P, and various concentrations of ADP and PEP were incubated at 37°C; the reactions were stopped at different time points with 10 l of 1 M HCl; and the reaction mixtures were incubated for 1 min and neutralized with NaOH. The reaction mixtures were diluted with 100 l acetonitrile, and the reactions were run at 25°C on a Waters I-class ultrahigh-performance liquid chromatograph (UHPLC) with a photodiode array (PDA) detector (258 nm) using an Acquity UHPLC ethylene-bridged hybrid (BEH) amide column (2.1 by 100 mm, 1.7 m). Gradient elution (delivered at 0.13 ml/min) was employed using 80/20 acrylamide-water with 0.1% NH 2 OH (A) and water with 0.1% NH 2 OH (B), which started at 90% A and decreased linearly to 55% A over 10 min.
Isothermal titration calorimetry. Binding interaction between OtsA and ligands was characterized at 25°C, using a MicroCal ITC200 titration calorimeter (MicroCal). An OtsA concentration of 60 M was used for all titrations. Ligands (1 mM) were injected in 2-l aliquots. Titration data were recorded in 20 mM Tris (pH 7.5)-500 mM NaCl. Data were analyzed by fitting a simple single-site model using Origin software (MicroCal).
Accession numbers. The coordinates and structure factors presented in this work have been deposited in the Protein Data Bank under accession codes 5JIJ, 5K41, 5K42, 5JIO, 5L3K, 5K5C, and 5K44.