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Catalysis byN-acetyl-D-glucosaminylphosphatidylinositol de-N-acetylase

(PIG-L) fromEntamoeba histolytica: New roles for conserved residues

Mohammad Ashraf#, Perinthottathil Sreejith

#, Usha Yadav and Sneha Sudha Komath

School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Running title: Catalytic mechanism ofE. histolyticaPIG-L

#Equal contributing authors

To whom correspondence should be addressed: Sneha Sudha Komath (sskomath@mail.jnu.ac.in)School of Life Sciences, Jawaharlal Nehru University, New Delhi- 110 067 India. Telephone: +91 11

26704502. E-mail: sskomath@mail.jnu.ac.in; sskomath@yahoo.com

Keywords:Glycosylphosphatidylinositol anchor biosynthesis; GPI biosynthesis, PIG-L, GlcNAc-PI de-

N-acetylase,E. histolytica

Background:E. histolyticaPIG-L is active evenin 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 Asp46 and His140

participate in a general acid-base pair

mechanism, unusual for de-N-acetylases.Conclusion: PIG-L of amoeba is significantlydifferent from mammalian PIG-L.Significance:Probable drug-target for selective

delivery.Summary: We showed previously that

Entamoeba histolytica PIG-L exhibits a novelmetal-independent albeit metal-stimulatedactivity. Using mutational and biochemical

analysis, here we identify Asp46 and His140 ofthe enzyme as being important for catalysis. Weshow that these mutations neither affect theglobal conformational of the enzyme nor alter itsmetal binding affinity. The defect in catalysis,

due to the mutations, is specifically due to aneffect on Vmax and not due to altered substrateaffinity (or Km). We propose a general acid-base

pair mechanism to explain our results.

The biosynthesis of the glycosylphosphatidyl

inositol (GPI1) anchor is an essential andubiquitous pathway in eukaryotes. GPI-anchored

proteins are known to be involved in infectionand virulence of many eukaryotic pathogens,

including Trypanosoma, Leishmania, Candidaand Entamoeba (1-4).

The GPI anchor is synthesized in the ERin a step-wise process and transferred as a wholeonto the C-terminal ends of proteins that possessthe 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 ofthe gene are known to be lethal in all eukaryotesstudied so far (7). Besides being essential, it isone of the few steps of the GPI biosynthetic

pathway that involves a single enzyme rather

than a multi-protein complex, and takes place onthe cytoplasmic face of the endoplasmicreticulum (ER). The enzyme shows species-specific variations as well and may therefore bean attractive target for pathogen-specific drugs

(8).

PIG-L of E. histolytica (EhPIG-L) has

been reported to be important for amoebicpathogenesis (4). In a previous report weshowed that unlike rat PIG-L and other knownde-N-acetylases, EhPIG-L is actually capable oflow activity in the absence of metal and the

catalysis is stimulated by divalent cations (9).We also showed that, unlike other known PIG-Lenzymes, EhPIG-L preferred divalent cations

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.427245The latest version is atJBC Papers in Press. Published on January 22, 2013 as Manuscript M112.427245

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.427245http://www.jbc.org/cgi/doi/10.1074/jbc.M112.427245http://www.jbc.org/cgi/doi/10.1074/jbc.M112.427245http://www.jbc.org/cgi/doi/10.1074/jbc.M112.427245

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such a Mg2+, Mn2+or Co2+rather than Zn2+. Alsounusual, was the fact that the enzyme had anoptimum pH of 5.5. Interestingly, this pH wasnot altered by the presence or absence of

externally added metal, suggesting that the metaldid not play a direct role in catalysis as has been

proposed for other deacetylases. We alsoshowed that the role of the metal appeared to bein altering catalytic rates by inducing a

catalytically efficient conformation of theenzyme rather than in altering the affinity of theenzyme for its substrate. Thus, metal alteredVmax rather than the Km of the enzyme for itssubstrate (9).

All in all, it would appear that EhPIG-Lhas a significantly different catalytic pocket

from those described so far. We wondered

whether conserved residues within the catalyticpocket may also have taken on new functions inthe EhPIG-L enzyme as compared to the enzymefrom other sources. Therefore, in this report we

investigated the role of conserved aspartate andhistidine residues in the activity of the protein.

As before, we used the cytoplasmic catalyticdomain ofE. histolyticaPIG-L (EhTMPIG-L).

We show here that residues Asp46 and His140within the putative catalytic pocket areimportant for the activity of EhTMPIG-L and

provide a probable model for the catalysis.

EXPERIMENTAL PROCEDURESMaterials: The YPH-501 yeast strain was

procured from Institute of Microbial Technology

(Chandigrah, India) and DH5 cells fromBangalore Genei. UDP[6 3H]GlcNAc and acetic

anhydride were procured from Sigma (U.S.A),amylose resin from New England Biolabs(NEB) and Factor-Xa from Novagen. The

restriction enzymes and DNA polymerases werepurchased either from Bangalore Genei, MBI

Fermentas, or NEB. All other materials werepurchased either from Merck, Qualigens orSisco research laboratories.

Creation of the site-specific mutants: Usingprimers carrying site-specific mutations

(Supplemental Table I)2we amplifiedthe vector,

pMALEhTMPIG-L (pMAL-c2X plasmidbearing EhTMPIG-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 andused to transform DH5 cells. Colonies obtainedafter transformation were screened by colonyPCR using gene specific primers. The mutations

were further confirmed by DNA sequencing.

Expression and purification of MBP tagged

proteins:TB1 strain of E.coliwas transformedwith pMALEhTMPIG-L (which expressesEhTMPIG-L with a MBP tag at the N-

terminus) or its mutant variants and grown at37C to an OD 600nmof 0.5-0.6 in Luria-Bertanimedium containing 0.3 % (w/v) glucose. Proteinexpression and purification was carried outessentially as described previously (9). Briefly,

protein expression was induced with 0.25mMIPTG and the cells grown at 16C for another 16

hours. The proteins were affinity-purified fromamylose beads and used without removal of the

MBP-tag for all the enzyme assays. We havepreviously shown that the MBP-tag does notsignificantly alter the activity of the enzyme (9).

Assays for GlcNAc-PI de-N-acetylationactivity: The substrate for the assays was

prepared by exogenously providing UDP-[6 3H]-

N-acetylglucosamine (UDP-[6 3H] GlcNAc) toyeast (YPH-501) microsomes, as previously

described (9). This generally results in transferof [6 3H] GlcNAc from the donor, UDP-[6 3H]GlcNAc, to phosphatidylinositol (PI) by the

GPI-N-acetylglucosaminyltransferase (GPI-

GnT) enzyme involved in the first step of GPIbiosynthesis. Normally a significant amount ofthis desired substrate is also de-N-acetylated to[6 3H] glucosaminyl-PI ([6 3H] GlcN-PI) by the

endogenously present yeast PIG-L (GPI12) inthe microsomes. Therefore, for our assays, the [63H] GlcN-PI generated was reacetylated back

using acetic anhydride in order to provide uswith 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, thedried glycolipids containing [6 3H] GlcNAc-PIwere resuspended in 20l of acetate buffer (pH5.5) containing 50mM KCl, 10mM MgCl2,

10mM MnCl2. For enzyme assays carried out inthe absence of metal, MgCl2 and MnCl2 were

left out from the assay mixture. It is possible thatthe enzyme picks up some amount of metal fromthe cellular environment. However, as we show

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in this manuscript, the enzyme and its mutantscontinue to be able to bind to externally addedmetal and show roughly similar Kd values fordivalent metal, suggesting that the intrinsic

bound-metal, if any, is not very high in ourenzyme preparations. Approximately 4g of

protein was added to the lipid suspension andmixed gently by vortexing. This was thensonicated briefly and incubated at 37C for 2

hours in a total reaction volume of 40l. Theglycolipids were extracted in water-staurated

butanol, dried and resuspended in 10l of thesame solvent before being resolved on HPTLC

plates and analyzed by BioScan AR2000 TLC

scanner. For the steady state assays, the substratewas quantified by plotting a standard curve

using different known amounts of UDP[63H]GlcNAc as previously described (9). The

endogenously present unlabelled GlcNAc-PI inthe assay is not estimated by this method. So theKM and Vmax values for the catalytic activity

correspond to apparent rather than absolutevalues. However, the method is valid forcomparative analysis.

Far-UV circular dichroism (CD)spectroscopy: For CD spectroscopic studies, theMBP tag on the protein was cleaved withFactor-Xa followed by dialysis and passage

through amylose column to remove free MBP as

described previously (9). The far-UV CD spectra(average of 3 scans) of the wild type and mutantvariants (~0.03mg/ml in 10 mM acetate buffer,

pH 5.5, with 200mM NaCl, 10% glycerol) were

recorded between 260-200 nm at 25C in 1mmpathlength cuvette as previously described (9).

RESULTSPIG-L proteins belong to the family of metal-

dependent deacetylases. A sequence alignmentwith over a 100 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 andHXXH (10). Structural and biochemical studies

too pointed to the importance of the aspartateand histidine residues of these conserved motifsin metal binding and catalysis by different

metal-dependent deacetylases (10-12). In aprevious study we showed that EhPIG-L too

possesses an AHADD motif along with a HPNHmotif corresponding to these conserved motifs(9). Studying the role of the histidine andaspartate residues of these two motifs, therefore,

seemed to be a good starting point for ouranalysis.

Site directed mutagenesis: Using site directedmutagenesis, we mutated the conserved histidineand aspartate residues of the AHADD and

HXXH motifs to alanine to generate the mutantsEhTMPIG-L H43A, EhTMPIG-L D45A,EhTMPIG-L D46A, EhTMPIG-L H140Aand EhTMPIG-L H143A (Fig. 1A). In additionwe generated the EhTMPIG-L D47A mutant

(Fig. 1A). The most common residue at thisposition in eukaryotic GlcNAc-PI de-N-

acetylases is glutamate, but in other closehomologs there is considerable variability at this

position (10). Further, in order to probe the roleof other negatively charged residues in metal

binding, we mutated three other conserved

acidic residues to alanine to generate the mutantsEhTMPIG-L E79A, EhTMPIG-L D102A andEhTMPIG-L D133A (Fig. 1A). The MBP-

tagged mutant proteins were affinity-purifiedusing an amylose column by the protocol

described previously (9) (data not shown).

Secondary conformation of the mutantproteins: In order to ascertain whether the

specific site-directed mutants significantly

affected the global conformation of the protein,we carried out far-UV CD spectroscopic studieson the mutant variants of the protein afterremoval of the MBP tag. We observed that the

mutations did not significantly alter the globalconformations of the proteins (Fig. 1B).

Catalytic activity of the mutants and

identification of residues important foractivity: In order to test whether the specific

site-directed mutations significantly affected theenzymatic activity of the protein, we carried out

GlcNAc-PI de-N-acetylase activity assays both

in the absence and presence of externally addedmetal (Fig. 1C). It must be noted that theaddition of divalent cations stimulates thecatalytic activity of the wild type (WT) enzyme

by roughly 1.6 fold as compared to activity inthe absence of metal. The mutant proteins

possessed varying amounts of activity. Weclassified them into three major groups based on

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the catalytic activity that they exhibited (TableI).

The first group (Class I) comprised ofmutants that possessed ~60% activity or higher

even in the absence of externally added metaland were regarded as mutants that show no

significant impairment in catalytic activity.These included the mutant variants EhTMPIG-L E79A, EhTMPIG-L D102A, EhTMPIG-L

D133A as well as EhTMPIG-L D45A. Thus,Asp45, Glu79, Asp102, Asp133 were assumedto be relatively unimportant for the catalyticactivity of EhTMPIG-L.

In the second group (Class II), we placed

those mutants that showed significantly impairedcatalytic activity in the absence of added metal,

but whose activity could be stimulated by theaddition of metal. These included the mutants

EhTMPIG-L H43A, EhTMPIG-L D47A andEhTMPIG-L H143A. Of these, bothEhTMPIG-L H43A and EhTMPIG-L H143A

showed only ~20% of the activity of the wildtype EhTMPIG-L in the absence of externallyadded metal. However, EhTMPIG-L H43A

showed a roughly 6-fold stimulation in catalyticactivity upon addition of metal while

EhTMPIG-L H143A showed a 5-foldenhancement in activity under similarconditions. Thus, the catalytically active

conformation of the enzyme is attained in both

these mutants upon the addition of metal. Inother words, these mutants are impaired only inattainment of the catalytic conformation in theabsence of added metal. Thus, both His43 and

His143 appear to be important for the integrityof the active site conformation but not for metal

binding or catalysis itself. In comparison tothese mutants, EhTMPIG-L D47A showed ahigher level of activity in the absence of added

metal (34%). This mutant too was wellstimulated (2-fold) by the addition of metal.

Thus, Asp47 also does not appear to be a

catalytic residue.In the third group (Class III) we classified

those mutants that were significantly impaired incatalytic activity both in the absence of

externally added metal and upon addition ofmetal. The mutants EhTMPIG-L D46A and

EhTMPIG-L H140A belonged to this category.In order to determine whether Asp46 and His140could be catalytic residues, we investigated

metal binding as well as the steady state kineticparameters for the de-N-acetylation of [6 3H]GlcNAc-PI by these mutants, as described

below.

Metal binding by the mutant proteins: Wehave previously shown that binding of Mn2+ to

EhTMPIG-L results in a significant alterationin the global conformation of the protein thatcould be monitored by far-UV CD spectroscopy

(9). We therefore used CD spectroscopy tomonitor whether EhTMPIG-L D46A andEhTMPIG-L H140A had significantly alteredmetal binding. For comparison, we alsoanalyzed mutants from Class I and Class II. All

the mutants showed roughly similar extents ofglobal conformational changes on the addition

of metal as compared to the wild type (Fig. 2A).From the changes in the CD signal at

220nm upon titration with MnCl2, we obtainedbinding plots (Fig. 2B) and the dissociationconstants (Fig. 2B) for metal binding by the

different mutant variants. None of the mutants,including EhTMPIG-L D46A andEhTMPIG-L H140A, showed drastically

altered metal affinities. Given that divalentcations like Mn2+ prefer to form hexa-

coordinated complexes, it is possible thatmutation of a single residue in EhTMPIG-Ldoes not drastically affect metal affinities. Taken

together, it appears that the mutations do not

significantly alter either the enzymes ability tobind metal or its ability to undergo a globalconformational change upon metal binding.

Steady state analysis of the de-N-acetylaseactivity of the mutants: We next assessedwhether Kmor Vmaxvalues for GlcNAc-PI de-N-

acetylation were altered in the mutants,EhTMPIG-L D46A and EhTMPIG-LH140A, which could help explain the loss in

catalytic activity in these mutants. For thepurpose of comparison we also used the

EhTMPIG-L H43A mutant that had low

catalytic activity to begin with but was stronglystimulated by the addition of metal.

We have previously reported thatEhTMPIG-L shows a significantly higher Vmax

on the addition of metal but no alteration in theaffinity (or Km) for GlcNAc-PI (9). As can be

seen from Table II, the EhTMPIG-L H43Amutant showed no difference in affinity for thesubstrate (or Km) as compared to EhTMPIG-L,

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monitored in a parallel assay, in the absence aswell as presence of externally added metal. Butthe mutant did have lower Vmax values in theabsence of added metal, which explains the

lower activity observed in Fig. 1C as well. Theaddition of metal stimulates the activity by

enhancing the Vmaxof the reaction by roughly 2-fold and was statistically significant (p-valuevis--vis the assay in the absence of added metal

was 0.0021). In the parallel assay for the wild-type EhTMPIG-L also, a roughly two-foldenhancement in Vmax values in the presence ofmetal was observed, which was statisticallysignificant (p-value = 0.0012 when calculated

vis--vis the assay done in the absence of addedmetal) (Table II).

The mutants, EhTMPIG-L D46A andEhTMPIG-L H140A, on the other hand, had

low catalytic activity in the absence of metal.The Vmax values of both mutants were roughlyhalf that of the wild type EhTMPIG-L.

Additionally, both mutants were poorlystimulated by metal. As can be seen from TableII, the Vmax values were only marginally

improved by the addition of metal. Thestimulation observed upon addition of metal for

EhTMPIG-L D46A and EhTMPIG-L H140Awere not statistically significant (p-value = 0.26and 0.46, respectively, when calculated vis--vis

the assay carried out in the absence of added

metal in each case). The Km values, however,were not significantly affected in either of thesemutants in comparison to the wild type, both inthe absence or presence of externally added

metal, indicating that the binding of the substratewas likely to be largely unaffected by the

mutations.Thus, taken together, our results suggest

that both Asp46 and His140 are catalytic

residues in EhTMPIG-L.The proposed model: There are at least two

possible models that could be proposed to

explain the above results. For example, it ispossible to speculate that Asp46 and His140 arecritical for attainment of the catalyticallyefficient conformation of the active site. In the

absence of either Asp46 or His140, despitemetal binding and induction of the requisite

global conformational change, it is possible thatthe optimum geometry of the active site remainsunattained. Alternatively, it is possible to

speculate that Asp46 and His140 participate as ageneral acid-base pair (GABP) (Fig. 3),

somewhat like that suggested for LpxC, a Zn2+-

dependent UDP-3-O-((R)-3-hydroxymyristoyl)-

N-acetylglucosamine deacetylase but without thepolarization of a water molecule by the metal

(13).In such a GABP model, the deprotonated

Asp46 polarizes a molecule of water, generating

the nucleophile for attack on the carbonyl groupof the substrate. The intermediate that is thusformed is stabilized by the protonated His140. Inthe second step, the protonation of His140 byanother molecule of H2O promotes the

subsequent bond rearrangements that in turnassist the removal of the acetyl group from the

substrate.Such a model would also explain the low

level of activity observed in the mutantsEhTMPIG-L D46A and EhTMPIG-LH140A. Since substrate binding is unaffected,

we may assume that the substrate is sittingcorrectly in the pocket. Even in the absence ofHis140, in the EhTMPIG-L H140A mutant,

the nucleophile for attack on the amide bond iscreated by Asp46. But, in the absence of

stabilization of the intermediate and assistanceby His140, the probability of the acetyl groupleaving from the substrate is low, resulting in

much lower catalytic efficiency. Similarly, H2O

is a weak nucleophile in the absence of thepolarizing Asp46 in the EhTMPIG-L D46Amutant. Hence, the attack by a H2O moleculeoccurs with much lower probability in the

absence of Asp46. The protonated His140 wouldcontinue to stabilize the catalytic intermediate

and participate in the elimination of the acetylgroup. The low probability of attack by a watermolecule in the absence of the polarizing Asp46

would explain the much lower activity seen inthe EhTMPIG-L D46A mutant. Thus, absence

of either Asp46 or His140, in such a model,

would result in crippled, but not completelyabrogated, catalytic activity and metal-bindingwould be unable to compensate for the absenceof the key residue.

The major support to such a model alsocomes from the fact that the optimum pH for the

activity of EhTMPIG-L is 5.5. A deprotonatedaspartate with pKa ~4.5 and a protonatedhistidine with pKa of ~6.5 could participate to

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provide an optimum pH of 5.5 for the catalysis.In such a case, one would expect the optimum

pH of the mutant, EhTMPIG-L D46A, to shiftto a higher pH.

We tested this hypothesis by studying thepH profile of the de-N-acetylase activity of the

EhTMPIG-L D46A mutant. Indeed, the pHoptimum for this mutant was 6.5 as against 5.5for the wild type mutant (Fig. 4), lending

credence to a catalytic model that involves aGABP mechanism (Fig. 3). As a corollary tothis, we also expected that the optimum pH ofthe EhTMPIG-L H140 mutant would shift to

pH 4.5. However, due to issues of stability of the

EhTMPIG-L H140 mutant at low pH we wereunable to test whether the optimum pH for this

mutant had indeed shifted to the lower pH.

DISCUSSIONBased on homology and the presence of theconserved AHADD as well as HXXH motifs,

the eukaryotic PIG-L protein has been classifiedas a member of the larger family of metal-dependent deacetylases. This family of enzymes

includes, for example, MshB, a deacetylaseinvolved in mycothiol biosynthesis of M.

tuberculosis. The crystal structure of MshBprovided the first evidence for the role ofhistidine and aspartate residues of the AHADD

and HXXH motifs in metal ion co-ordination.

Baker and co-workers showed that His13 andAsp16 of the AHADD motif, along with the C-terminal His147 of the HPDH motif, wereinvolved in co-ordinating the central Zn2+ in

MshB (11). The authors also suggested thatAsp15 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 tonucleophilic attack by a water molecule which,

in turn, had been polarized by Asp15. Building

on this model, Ferguson and co-workers usedsemi-quantitative complementation assays inconjunction with homology modeling to studythe metal-binding and catalytic residues of rat

PIG-L (12). From this data, they proposed a rolefor His49 and Asp52 of the AHPDD motif,along with the C-terminal His157 of a HSNHmotif, in metal binding. Based on its positioning

within the catalytic pocket and the fact that thiswas the only mutant that showed no activity in

their assays, they also hypothesized that Asp51of 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 thehomologous conserved motifs described above(9). However, presence of the metal ion is not

critical for the function of EhTMPIG-L and wehave shown previously that the metal ion alters

the Vmax but not the Km of the enzyme for itssubstrate (9). We show here that this is also the

case with the mutants of this enzyme,EhTMPIG-L H43A, EhTMPIG-L D46A andEhTMPIG-L H140A. In other words, the metal

ion plays no role in substrate binding byEhTMPIG-L. The metal ion also does notappear to polarize a water molecule, as has been

proposed for other deacetylases; if it did, itwould have lowered the pKaof water and hence

altered the optimum pH at which the enzymewould work (9). Thus, the mechanism ofcatalysis does not appear to be conserved.

Indeed, our results suggest that although the

AHADD and HXXH motifs continue to beimportant for the functioning of the PIG-Lenzyme from E. histolytica, the conservedresidues appear to have taken on new functions.

Specifically, Asp46 and His140, instead ofbinding to metal, as in other deacetylases, now

appear to participate directly in the catalysisitself, as a general acid-base pair. Thatconserved residues can take on new functions in

the course of evolution is certainly veryinteresting. But more interesting, perhaps, from

the clinical biochemistry point of view is the fact

that this suggests the possibility of selectivelytargeting the pathogen vis--vis the host byidentifying specific inhibitors to the E.histolyticaPIG-L.

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FOOTNOTESThis work was supported by a research grant from CSIR to SSK (No. 37(1441)/10/EMR-II). MA and UYthank CSIR for fellowships. The CD spectra were recorded at the Advanced Instrumentation ResearchFacility at JNU.

1Abbreviations:EhTMPIG-L,transmembrane-deletedEntamoeba histolyticaN-acetyl-D-

glucosaminylphosphatidylinositol de-N-acetylase; GPI, glycosylphosphatidyl inositol; GlcNAc-PI,N-acetylglucosaminylphosphatidylinositol; GlcN-PI, glucosaminylphosphatidylinositol2Supplemental Table I.

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FIGURE LEGENDS:

Fig. 1: Effect of mutations in EhTMPIG-L on the global conformation and activity of the enzyme.(A)A schematic representation of the different mutations introduced in the EhTMPIG-L. The deleted

putative TM domain is shown by dotted lines. (B)Far-UV CD spectra of EhTMPIG-L as well as itsdifferent point mutants showing that the mutations do not cause any major changes in global

conformation. WT in the figure refers to EhTMPIG-L; H43A, D45A, D46A, D47A, E79A, D102A,D133A, H140A, H143A refer to the specific mutations in the mutant variants of EhTMPIG-L. (C)GlcNAc-PI de-N-acetylase activity of EhTMPIG-L and its mutants in the absence and presence of

externally added metal.The de-N-acetylase activities were carried out at pH 5.5 and 37oC as described inthe text. A 2 hour time point was taken for the assay in the presence of metal and 4 hour time-point in theabsence of externally added metal. The data for the activity in the absence of externally added metal wasthen calculated for a two-hour time period, assuming linearity of the assay (9). The data is representedhere is normalized with respect to that observed for EhTMPIG-L in the presence of added metal.

Roughly 80% of the [6 3H] GlcNAc-PI was converted to [6 3H] GlcN-PI by EhTMPIG-L in the two-hour period in the presence of added metal.

Fig. 2:Metal binding is unaffected in the mutants of EhTMPIG-L. (A) The far UV spectra of WT

EhTMPIG-L and its mutants. The spectra in the absence of externally added metal (no metal) or inthe presence of externally added 3 M MnCl2 (+ metal)were recorded after removal of the MBP fusiontags as described in the text. (B) Binding plots showing affinity of EhTMPIG-L and its mutants forMn2+. The proteins were titrated with (0-3.0M MnCl2) and incubated at 25

oC for 5 minutes after each

addition before recording the CD spectra. Normalized changes in CD signal at 220nm were used to obtainthe binding plot for the proteins as a function of ligand concentration. The data was fit using Sigma Plot

8.0 assuming a one-site binding model. The average of two independent data sets done in duplicates wastaken for Kdestimation. Bmaxrepresents the value at saturation. R

2values corresponding to the goodness

of the fits ranged from 0.95 to 0.99. The data shown is average of two independent experiments done induplicates.

Fig. 3: The general acid-base pair (GABP) mechanism proposed for the catalytic activity of E.histolytica

PIG-L.The deprotonated Asp46 polarizes a molecule of water, generating the nucleophile forattack at the carbonyl moiety of the substrate in the first step. The protonated His140 stabilizes the

intermediate formed in this process, making a hydrogen bond with the substrate. In the second step, thesubsequent bond rearrangements result in cleavage of the acetyl group from the substrate and

simultaneous re-protonation of His140 through the participation of a second molecule of H2O.

Fig. 4: pH dependence of EhTMPIG-L D46A mutant versus the wild type and the proposedcatalytic model.The activity of the two protein variants was studied as a function of pH in 50mM acetate(pH 3.5, 4.5, 5.5, 6.5) or in 50mM HEPES (pH 7.5, 8.5) buffers as reported previously (9). The data

shown is for 2 hours in the absence of externally added divalent metal. The activity for each protein atdifferent pH is shown relative to the maximum activity (100%) exhibited by it.

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Table I: Classification and steady state kinetic parameters of EhTMPIG-L mutants.

On the basis of activity the mutants were classified into three groups.

Mutants Activity vis--vis EhTMPIG-L

Class-I EhTMPIG-L D45A

EhTMPIG-L E79A

EhTMPIG-L D102A

EhTMPIG-L D133A

good activity in absence of metal;

well stimulated upon addition of metal

Class-II EhTMPIG-L H43A

EhTMPIG-L D47A

EhTMPIG-L H143A

low activity in absence of metal;

well stimulated upon addition of metal

Class-III EhTMPIG-L D46A

EhTMPIG-L H140A

low activity in absence of metal;

no stimulation upon addition of metal

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Table II: Steady state parameters for de-N-acetylase activity of the wild type EhTMPIG-L versusthe mutants.The substrate (~ 1 nmole) was incubated at 37oC with ~ 4 g of the protein variants in theabsence orpresence of externally added metal in acetate buffer (pH 5.5) as previously reported (9). Asingle batch of pooled substrate was used for all assays to reduce errors due to varying levels of

endogenous unlabelled GlcNAc-PI from batch to batch. In absence of externally added metal, the assaywas carried out for 4 hours while in the presence of externally added metal it was done for 2 hours (the

enzyme activity is linear in this range); the velocity of the reaction (V) in terms of pmoles of productformed was monitored as a function of input substrate concentration (S) using BioScan AR2000 asreported previously (9). Lineweaver-Burk plots of V-1(pmoles.hr-1.g protein-1) versus S-1(M-1) were

plotted in order to determine the apparent Kmand Vmaxvalues. The data is average of two parallelexperiments done using different preparations of enzyme. The p-values shown in the table in each set(without or with added metal) are with reference to the data for the wild type protein under similar assayconditions. The differences in Kmvalues for mutants versus EhTMPIG-L, in absence or presence ofexternally added metal, were not statistically significant (in all cases, p-values > 0.1).

Sample No added metal With added metal

Km (M) Vmax

pmol.hr-1

.g protein-1

Km(M) Vmax

pmol.hr-1

.g protein-1

EhTMPIG-L 1.95 0.20 64.8 2.7 2.09 0.23 121.6 4.9

EhTMPIG-L

H43A

1.99 0.18 42.6 1.3

(p-value =0.018)

2.63 0.59 74.2 3.8

(p-value =0.017)

EhTMPIG-L

D46A

1.82 0.14 33.3 0.0

(p-value =0.007)

1.95 0.22 37.9 3.0

(p-value =0.005)

EhTMPIG-L

H140A

1.6 0.25 30.6 9.8

(p-value = 0.007)

1.37 0.21 40.3 4.6

(p-value =0.007)*Previously we reported EhTMPIG-L to have an apparent KMof 1.95 0.24 M and a Vmax of 55.15 2.96

pmol.hr-1

.g protein-1

in the absence of externally added metal; in the presence of externally added metal theapparent KMwas 2.05 0.18 M and Vmaxwas 130.08 5.62 pmol.hr

-1.g protein

-1(9). We attribute these

differences to experimental variations and variations in amount of endogenous unlabelled GlcNAc-PI/ GlcN-PIin the substrate preparations.

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Fig. 1

A.

B.

C.

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Fig. 2

A.

B.

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Fig. 3

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Fig. 4