Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica

Background: E. histolytica PIG-L is active even in absence of metal, unlike other homologs. Metal stimulation of activity alters Vmax, not Km. Metal does not alter optimum pH of catalysis. What explains these differences? Results: Conserved Asp-46 and His-140 participate in a general acid-base pair mechanism, unusual for de-N-acetylases. Conclusion: PIG-L of amoeba is significantly different from mammalian PIG-L. Significance: We have identified a probable drug target for selective delivery. We showed previously that Entamoeba histolytica PIG-L exhibits a novel metal-independent albeit metal-stimulated activity. Using mutational and biochemical analysis, here we identify Asp-46 and His-140 of the enzyme as being important for catalysis. We show that these mutations neither affect the global conformational of the enzyme nor alter its metal binding affinity. The defect in catalysis, due to the mutations, is specifically due to an effect on Vmax and not due to altered substrate affinity (or Km). We propose a general acid-base pair mechanism to explain our results.

The biosynthesis of the glycosylphosphatidylinositol (GPI) 3 anchor is an essential and ubiquitous pathway in eukaryotes. GPI-anchored proteins are known to be involved in infection and virulence of many eukaryotic pathogens, including Trypanosoma, Leishmania, Candida, and Entamoeba (1)(2)(3)(4).
The GPI anchor is synthesized in the endoplasmic reticulum in a stepwise process and transferred as a whole onto the C termini of proteins that possess the GPI-anchoring signal sequence (5). The PIG-L enzyme functions at the second step of GPI anchor biosynthesis, converting N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) to glucosaminylphosphatidylinositol (GlcN-PI) (5,6). This step is conserved in the GPI biosynthesis pathway, and deletion mutations of the gene are known to be lethal in all eukaryotes studied so far (7). Besides being essential, it is one of the few steps of the GPI biosynthetic pathway that involves a single enzyme rather than a multiprotein complex and takes place on the cytoplasmic face of the endoplasmic reticulum. The enzyme shows species-specific variations as well and may therefore be an attractive target for pathogen-specific drugs (8).
PIG-L of Entamoeba histolytica (EhPIG-L) has been reported to be important for amoebic pathogenesis (4). In a previous report we showed that unlike rat PIG-L and other known de-Nacetylases, EhPIG-L is actually capable of low activity in the absence of metal, and the catalysis is stimulated by divalent cations (9). We also showed that, unlike other known PIG-L enzymes, EhPIG-L preferred divalent cations such a Mg 2ϩ , Mn 2ϩ , or Co 2ϩ rather than Zn 2ϩ . Also unusual was the fact that the enzyme had an optimum pH of 5.5. Interestingly, this pH was not altered by the presence or absence of externally added metal, suggesting that the metal did not play a direct role in catalysis as has been proposed for other deacetylases. We also showed that the role of the metal appeared to be in altering catalytic rates by inducing a catalytically efficient conformation of the enzyme rather than in altering the affinity of the enzyme for its substrate. Thus, metal altered V max rather than the K m of the enzyme for its substrate (9).
All in all, it would appear that EhPIG-L has a significantly different catalytic pocket from those described so far. We wondered whether conserved residues within the catalytic pocket may also have taken on new functions in the EhPIG-L enzyme compared with the enzyme from other sources. Therefore, in this report we investigated the role of conserved aspartate and histidine residues in the activity of the protein. As before, we used the cytoplasmic catalytic domain of E. histolytica PIG-L (Eh⌬TMPIG-L). We show here that residues Asp-46 and His-140 within the putative catalytic pocket are important for the activity of Eh⌬TMPIG-L and provide a probable model for the catalysis.

EXPERIMENTAL PROCEDURES
Materials-The YPH-501 yeast strain was procured from Institute of Microbial Technology (Chandigrah, India) and DH5␣ cells from Bangalore Genei. UDP-[6-3 H]GlcNAc and acetic anhydride were procured from Sigma, amylose resin from New England Biolabs (NEB), and Factor Xa from Novagen. The restriction enzymes and DNA polymerases were purchased either from Bangalore Genei, MBI Fermentas, or NEB.
All other materials were purchased either from Merck, Qualigens, or Sisco research laboratories.
Creation of the Site-specific Mutants-Using primers carrying site-specific mutations (see supplemental Table 1) we amplified the vector, pMALEh⌬TMPIG-L (pMAL-c2X plasmid bearing Eh⌬TMPIG-L, a truncation mutant of full-length EhPIG-L lacking the first 24 N-terminal residues (9)). The PCR product was then digested by Dpn1 restriction enzyme and used to transform DH5␣ cells. Colonies obtained after transformation were screened by colony PCR using gene-specific primers. The mutations were further confirmed by DNA sequencing.
Expression and Purification of MBP-tagged Proteins-The TB1 strain of Escherichia coli was transformed with pMALEh⌬TMPIG-L (which expresses Eh⌬TMPIG-L with a MBP tag at the N terminus) or its mutant variants and grown at 37°C to an A 600 nm of 0.5-0.6 in Luria-Bertani medium containing 0.3% (w/v) glucose. Protein expression and purification were carried out essentially as described previously (9). Briefly, protein expression was induced with 0.25 mM isopropyl 1-thio-␤-D-galactopyranoside, and the cells were grown at 16°C for another 16 h. The proteins were affinity-purified from amylose beads and used without removal of the MBP tag for all the enzyme assays. We have previously shown that the MBP tag does not significantly alter the activity of the enzyme (9).
Assays for GlcNAc-PI De-N-acetylation Activity-The substrate for the assays was prepared by exogenously providing UDP-[6-3 H]N-acetylglucosamine (UDP-[6-3 H]GlcNAc) to yeast (YPH-501) microsomes, as described previously (9). This generally results in transfer of [6-3 H]GlcNAc from the donor, UDP-[6-3 H]GlcNAc, to phosphatidylinositol (PI) by the GPI-N-acetylglucosaminyltransferase enzyme involved in the first step of GPI biosynthesis. Normally a significant amount of this desired substrate is also de-N-acetylated to [6-3 H]glucosaminyl-PI ([6-3 H]GlcN-PI) by the endogenously present yeast PIG-L (GPI12) in the microsomes. Therefore, for our assays, the [6-3 H]GlcN-PI generated was reacetylated back using acetic anhydride to provide us with sufficient amount of the substrate for the assays (9).
The GlcNAc-PI de-N-acetylation assays were carried out with no modifications to our previously reported protocol (9). In brief, the dried glycolipids containing [6-3 H]GlcNAc-PI were resuspended in 20 l of acetate buffer, pH 5.5, containing 50 mM KCl, 10 mM MgCl 2 , 10 mM MnCl 2 . For enzyme assays carried out "in the absence of metal," MgCl 2 and MnCl 2 were left out from the assay mixture. It is possible that the enzyme picks up some amount of metal from the cellular environment. However, as we show in this paper, the enzyme and its mutants continue to be able to bind to externally added metal and show approximately similar K d values for divalent metal, suggesting that the intrinsic bound metal, if any, is not very high in our enzyme preparations. Approximately 4 g of protein was added to the lipid suspension and mixed gently by vortexing. This was then sonicated briefly and incubated at 37°C for 2 h in a total reaction volume of 40 l. The glycolipids were extracted in water-saturated butanol, dried, and resuspended in 10 l of the same solvent before being resolved on HPTLC plates and analyzed by BioScan AR2000 TLC scanner. For the steady-state assays, the substrate was quantified by plotting a standard curve using different known amounts of UDP[6-3 H]GlcNAc as described previously (9). The endogenously present unlabeled GlcNAc-PI in the assay is not estimated by this method. So the K m and V max values for the catalytic activity correspond to "apparent" rather than "absolute" values. However, the method is valid for comparative analysis.
Far-UV Circular Dichroism (CD) Spectroscopy-For CD spectroscopic studies, the MBP tag on the protein was cleaved with Factor Xa followed by dialysis and passage through amylose column to remove free MBP as described previously (9). The far-UV CD spectra (average of three scans) of the wild type and mutant variants (ϳ0.03 mg/ml in 10 mM acetate buffer, pH 5.5, with 200 mM NaCl, 10% glycerol) were recorded between 260 and 200 nm at 25°C in a 1-mm path length cuvette as described previously (9).

RESULTS
PIG-L proteins belong to the family of metal-dependent deacetylases. A sequence alignment with Ͼ100 homologous proteins from archaea, bacteria, protozoa, and other eukaryotes, including PIG-L from mammals, suggested the presence of two conserved motifs with the consensus sequences (P/A)-H-(P/A)-DD and HXXH (10). Structural and biochemical studies too pointed to the importance of the aspartate and histidine residues of these conserved motifs in metal binding and catalysis by different metal-dependent deacetylases (10 -12). In a previous study we showed that EhPIG-L too possesses an AHADD motif along with a HPNH motif corresponding to these conserved motifs (9). Studying the role of the histidine and aspartate residues of these two motifs, therefore, seemed to be a good starting point for our analysis.
Site-directed Mutagenesis-Using site-directed mutagenesis, we mutated the conserved histidine and aspartate residues of the AHADD and HXXH motifs to alanine to generate the mutants Eh⌬TMPIG-L H43A, Eh⌬TMPIG-L D45A, Eh⌬TMPIG-L D46A, Eh⌬TMPIG-L H140A, and Eh⌬TMPIG-L H143A (Fig. 1A). In addition we generated the Eh⌬TMPIG-L D47A mutant (Fig. 1A). The most common residue at this position in eukaryotic GlcNAc-PI de-N-acetylases is glutamate, but in other close homologs there is considerable variability at this position (10). Further, to probe the role of other negatively charged residues in metal binding, we mutated three other conserved acidic residues to alanine to generate the mutants Eh⌬TMPIG-L E79A, Eh⌬TMPIG-L D102A, and Eh⌬TMPIG-L D133A (Fig. 1A). The MBP-tagged mutant proteins were affinity-purified using an amylose column by the protocol described previously (9) (data not shown).
Secondary Conformation of the Mutant Proteins-To ascertain whether the specific site-directed mutants significantly affected the global conformation of the protein, we carried out far-UV CD spectroscopic studies on the mutant variants of the protein after removal of the MBP tag. We observed that the mutations did not significantly alter the global conformations of the proteins (Fig. 1B).
Catalytic Activity of the Mutants and Identification of Residues Important for Activity-To test whether the specific sitedirected mutations significantly affected the enzymatic activity of the protein, we carried out GlcNAc-PI de-N-acetylase activity assays both in the absence and presence of externally added metal (Fig. 1C). It must be noted that the addition of divalent cations stimulates the catalytic activity of the wild type (WT) enzyme by approximately 1.6-fold compared with activity in the absence of metal. The mutant proteins possessed varying amounts of activity. We classified them into three major groups based on the catalytic activity that they exhibited ( Table 1).
The first group (Class I) comprised mutants that possessed ϳ60% activity or higher even in the absence of externally added metal and were regarded as mutants that show no significant impairment in catalytic activity. These included the mutant variants Eh⌬TMPIG-L E79A, Eh⌬TMPIG-L D102A, Eh⌬TMPIG-L D133A, and Eh⌬TMPIG-L D45A. Thus, Asp-45, Glu-79, Asp-102, and Asp-133 were assumed to be relatively unimportant for the catalytic activity of Eh⌬TMPIG-L.
In the second group (Class II), we placed those mutants that showed significantly impaired catalytic activity in the absence of added metal, but whose activity could be stimulated by the addition of metal. These included the mutants Eh⌬TMPIG-L H43A, Eh⌬TMPIG-L D47A, and Eh⌬TMPIG-L H143A. Of these, both Eh⌬TMPIG-L H43A and Eh⌬TMPIG-L H143A showed only ϳ20% of the activity of the wild type Eh⌬TMPIG-L in the absence of externally added metal. However, Eh⌬TMPIG-L H43A showed an approximately 6-fold stimulation in catalytic activity upon addition of metal whereas Eh⌬TMPIG-L H143A showed a 5-fold enhancement in activity under similar conditions. Thus, the catalytically active conformation of the enzyme is attained in both these mutants upon the addition of metal. In other words, these mutants are impaired only in attainment of the catalytic conformation in the absence of added metal. Thus, both His-43 and His-143 appear to be important for the integrity of the active site conformation but not for metal binding or catalysis itself. In comparison to these mutants, Eh⌬TMPIG-L D47A showed a higher level of activity in the absence of added metal (34%). This mutant too was well stimulated (2-fold) by the addition of metal. Thus, Asp-47 also does not appear to be a catalytic residue.
In the third group (Class III) we placed those mutants that were significantly impaired in catalytic activity both in the absence of externally added metal and upon addition of metal. The mutants Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A belonged to this category. To determine whether Asp-46 and His-140 could be catalytic residues, we investigated metal binding as well as the steady-state kinetic parameters for the de-Nacetylation of [6-3 H]GlcNAc-PI by these mutants, as described below.
Metal Binding by the Mutant Proteins-We have previously shown that binding of Mn 2ϩ to Eh⌬TMPIG-L results in a significant alteration in the global conformation of the protein that could be monitored by far-UV CD spectroscopy (9). We therefore used CD spectroscopy to monitor whether Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A had significantly altered metal binding. For comparison, we also analyzed mutants from Class I and Class II. All of the mutants showed approximately similar extents of global conformational

TABLE 1 Classification and steady-state kinetic parameters of Eh⌬TMPIG-L mutants
changes on the addition of metal compared with the wild type ( Fig. 2A).
From the changes in the CD signal at 220 nm upon titration with MnCl 2 , we obtained binding plots (Fig. 2B) and the dissociation constants (Fig. 2B) for metal binding by the different mutant variants. None of the mutants, including Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A, showed drastically altered metal affinities. Given that divalent cations like Mn 2ϩ prefer to form hexa-coordinated complexes, it is possible that mutation of a single residue in Eh⌬TMPIG-L does not drastically affect metal affinities. Taken together, it appears that the mutations do not significantly alter the ability of either enzyme to bind metal or its ability to undergo a global conformational change upon metal binding.
Steady-state Analysis of the De-N-acetylase Activity of the Mutants-We next assessed whether K m or V max values for GlcNAc-PI de-N-acetylation were altered in the mutants Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A, which could help explain the loss in catalytic activity in these mutants. For the purpose of comparison we also used the Eh⌬TMPIG-L H43A mutant that had low catalytic activity to begin with but was strongly stimulated by the addition of metal.
We have previously reported that Eh⌬TMPIG-L shows a significantly higher V max on the addition of metal but no alteration in the affinity (or K m ) for GlcNAc-PI (9). As can be seen from Table 2, the Eh⌬TMPIG-L H43A mutant showed no difference in affinity for the substrate (or K m ) compared with Eh⌬TMPIG-L, monitored in a parallel assay, in the absence as well as presence of externally added metal. But the mutant did have lower V max values in the absence of added metal, which explains the lower activity observed in Fig. 1C as well. The addition of metal stimulates the activity by enhancing the V max of the reaction by approximately 2-fold and was statistically significant (p value vis-à-vis the assay in the absence of added metal was 0.0021). In the parallel assay for the wild type Eh⌬TMPIG-L also, an approximately 2-fold enhancement in V max values in the presence of metal was observed, which was statistically significant (p ϭ 0.0012 when calculated vis-à-vis the assay done in the absence of added metal) ( Table 2).
The mutants Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A on the other hand, had low catalytic activity in the absence of metal. The V max values of both mutants were approximately half that of the wild type Eh⌬TMPIG-L. Additionally, both mutants were poorly stimulated by metal. As can be seen from Table 2, the V max values were only marginally improved by the addition of metal. The stimulation observed upon addition of metal for Eh⌬TMPIG-L D46A and Eh⌬TMPIG-L H140A was not statistically significant (p ϭ 0.26 and 0.46, respectively, when calculated vis-à-vis the assay carried out in the absence of added metal in each case). The K m values, however, were not significantly affected in either of these mutants compared with the wild type, both in the absence or presence of externally added metal, indicating that the binding of the substrate was likely to be largely unaffected by the mutations. Thus, taken together, our results suggest that both Asp-46 and His-140 are catalytic residues in Eh⌬TMPIG-L.
The Proposed Model-There are at least two possible models that could be proposed to explain the above results. For example, it is possible to speculate that Asp-46 and His-140 are critical for attainment of the catalytically efficient conformation of the active site. In the absence of either Asp-46 or His-140, despite metal binding and induction of the requisite global conformational change, it is possible that the optimum geometry of the active site remains unattained. Alternatively, it is possible to speculate that Asp-46 and His-140 participate as a general acidbase pair (GABP) (Fig. 3), somewhat like that suggested for LpxC, a Zn 2ϩ -dependent UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase but without the polarization of a water molecule by the metal (13).
In such a general acid-base pair model, the deprotonated Asp-46 polarizes a molecule of water, generating the nucleophile for attack on the carbonyl group of the substrate. The intermediate that is thus formed is stabilized by the protonated His-140. In the second step, the protonation of His-140 by another molecule of H 2 O promotes the subsequent bond rearrangements that in turn assist the removal of the acetyl group from the substrate.
Such a model would also explain the low level of activity observed in the mutants EhPIG-L⌬TM D46A and EhPIG-L⌬TM H140A. Because substrate binding is unaffected, we may assume that the substrate is sitting correctly in the pocket. Even in the absence of His-140, in the EhPIG-L⌬TM H140A mutant, the nucleophile for attack on the amide bond is created by Asp-46; but, in the absence of stabilization of the intermediate and and incubated at 25°C for 5 min after each addition before recording the CD spectra. Normalized changes in CD signal at 220 nm were used to obtain the binding plot for the proteins as a function of ligand concentration. The data were fit using Sigma Plot 8.0 assuming a one-site binding model. The average of two independent data sets done in duplicate was taken for K d estimation. B max represents the value at saturation. R 2 values corresponding to the goodness of the fits ranged from 0.95 to 0.99. The data shown are the average of two independent experiments done in duplicate.

Catalytic Mechanism of E. histolytica PIG-L
MARCH 15, 2013 • VOLUME 288 • NUMBER 11 assistance by His-140, the probability of the acetyl group leaving from the substrate is low, resulting in much lower catalytic efficiency. Similarly, H 2 O is a weak nucleophile in the absence of the polarizing Asp-46 in the EhPIG-L⌬TM D46A mutant. Hence, the attack by a H 2 O molecule occurs with much lower probability in the absence of Asp-46. The protonated His-140 would continue to stabilize the catalytic intermediate and participate in the elimination of the acetyl group. The low probability of attack by a water molecule in the absence of the polarizing Asp-46 would explain the much lower activity seen in the EhPIG-L⌬TM D46A mutant. Thus, the absence of either Asp-46 or His-140 in such a model would result in crippled, but not completely abrogated, catalytic activity, and metal binding would be unable to compensate for the absence of the key residue.
The major support for such a model also comes from the fact that the optimum pH for the activity of Eh⌬TMPIG-L is 5.5. A deprotonated aspartate with pK a ϳ4.5 and a protonated histidine with pK a of ϳ6.5 could participate to provide an optimum pH of 5.5 for the catalysis. In such a case, one would expect the optimum pH of the mutant, Eh⌬TMPIG-L D46A, to shift to a higher pH.
We tested this hypothesis by studying the pH profile of the de-N-acetylase activity of the Eh⌬TMPIG-L D46A mutant. Indeed, the pH optimum for this mutant was 6.5 as against 5.5 for the wild type mutant (Fig. 4), lending credence to a catalytic model that involves a general acid-base pair mechanism. As a corollary to this, we also expected that the optimum pH of the Eh⌬TMPIG-L H140 mutant would shift to pH 4.5. However, due to issues of stability of the Eh⌬TMPIG-L H140 mutant at low pH we were unable to test whether the optimum pH for this mutant had indeed shifted to the lower pH.

DISCUSSION
Based on homology and the presence of the conserved AHADD as well as HXXH motifs, the eukaryotic PIG-L protein has been classified as a member of the larger family of metaldependent deacetylases. This family of enzymes includes, for example, MshB, a deacetylase involved in mycothiol biosynthesis of Mycobacterium tuberculosis. The crystal structure of MshB provided the first evidence for the role of histidine and aspartate residues of the AHADD and HXXH motifs in metal ion co-ordination. Baker and co-workers showed that His-13 and Asp-16 of the AHADD motif, along with the C-terminal His-147 of the HPDH motif, were involved in co-ordinating the central Zn 2ϩ in MshB (11). The authors also suggested that Asp-15 of the AHADD motif was ideally placed in the catalytic pocket to act as a catalytic base. They proposed a model in which the carbonyl bond of the substrate was polarized by the central metal, making it susceptible to nucleophilic

Steady-state parameters for de-N-acetylase activity of the wild type Eh⌬TMPIG-L versus the mutants
The substrate (ϳ1 nmol) was incubated at 37°C with ϳ4 g of the protein variants in the absence or presence of externally added metal in acetate buffer (pH 5.5) as reported previously (9). A single batch of pooled substrate was used for all assays to reduce errors due to varying levels of endogenous unlabeled GlcNAc-PI from batch to batch. In absence of externally added metal, the assay was carried out for 4 h, whereas in the presence of externally added metal it was done for 2 h (the enzyme activity is linear in this range); the velocity of the reaction (V) in terms of pmoles of product formed was monitored as a function of input substrate concentration (S) using BioScan AR2000 as reported previously (9). Lineweaver-Burk plots of V Ϫ1 (pmol⅐h Ϫ1 per g of protein) versus S Ϫ1 (M Ϫ1 ) were plotted to determine the apparent K m and V max values. The data are the average of two parallel experiments done using different preparations of enzyme. The p values shown in the table in each set (with or without added metal) are with reference to the data for the wild type protein under similar assay conditions. The differences in K m values for mutants versus Eh⌬TMPIG-L, in the absence or presence of externally added metal, were not statistically significant (in all cases, p values Ͼ 0.1).

No added metal
With added metal   The activity of the two protein variants was studied as a function of pH in 50 mM acetate (pH 3.5, 4.5, 5.5, 6.5) or in 50 mM HEPES (pH 7.5, 8.5) buffers as reported previously (9). The data shown are for 2 h in the absence of externally added divalent metal. The activity for each protein at different pH is shown relative to the maximum activity (100%) exhibited by it.
attack by a water molecule which, in turn, had been polarized by Asp-15. Building on this model, Ferguson and co-workers used semi-quantitative complementation assays in conjunction with homology modeling to study the metal-binding and catalytic residues of rat PIG-L (12). From this data, they proposed a role for His-49 and Asp-52 of the AHPDD motif, along with the C-terminal His-157 of a HSNH motif, in metal binding. Based on its positioning within the catalytic pocket and the fact that this was the only mutant that showed no activity in their assays, they also hypothesized that Asp-51 of the AHPDD motif could act as a catalytic base and proposed a catalytic model very similar to the one proposed for MshB by Baker and co-workers. PIG-L from E. histolytica too has the homologous conserved motifs described above (9). However, presence of the metal ion is not critical for the function of Eh⌬TMPIG-L, and we have shown previously that the metal ion alters the V max but not the K m of the enzyme for its substrate (9). We show here that this is also the case with the mutants of this enzyme, Eh⌬TMPIG-L H43A, Eh⌬TMPIG-L D46A, and Eh⌬TMPIG-L H140A. In other words, the metal ion plays no role in substrate binding by Eh⌬TMPIG-L. The metal ion also does not appear to polarize a water molecule, as has been proposed for other deacetylases; if it did, it would have lowered the pK a of water and hence altered the optimum pH at which the enzyme would work (9). Thus, the mechanism of catalysis does not appear to be conserved. Indeed, our results suggest that although the AHADD and HXXH motifs continue to be important for the functioning of the PIG-L enzyme from E. histolytica, the conserved residues appear to have taken on new functions. Specifically, Asp-46 and His-140, instead of binding to metal, as in other deacetylases, now appear to participate directly in the catalysis itself, as a general acid-base pair. That conserved residues can take on new functions in the course of evolution is certainly very interesting. But more interesting, perhaps, from the clinical biochemistry point of view is the fact that this suggests the possibility of selectively targeting the pathogen vis-à-vis the host by identifying specific inhibitors to the E. histolytica PIG-L.