Crystal structure of calcium binding protein-5 from Entamoeba histolytica and its involvement in initiation of phagocytosis of human erythrocytes.

Entamoeba histolytica is the etiological agent of human amoebic colitis and liver abscess, and causes a high level of morbidity and mortality worldwide, particularly in developing countries. There are a number of studies that have shown a crucial role for Ca2+ and its binding protein in amoebic biology. EhCaBP5 is one of the EF hand calcium-binding proteins of E. histolytica. We have determined the crystal structure of EhCaBP5 at 1.9 Šresolution in the Ca2+-bound state, which shows an unconventional mode of Ca2+ binding involving coordination to a closed yet canonical EF-hand motif. Structurally, EhCaBP5 is more similar to the essential light chain of myosin than to Calmodulin despite its somewhat greater sequence identity with Calmodulin. This structure-based analysis suggests that EhCaBP5 could be a light chain of myosin. Surface plasmon resonance studies confirmed this hypothesis, and in particular showed that EhCaBP5 interacts with the IQ motif of myosin 1B in calcium independent manner. It also appears from modelling of the EhCaBP5-IQ motif complex that EhCaBP5 undergoes a structural change in order to bind the IQ motif of myosin. This specific interaction was further confirmed by the observation that EhCaBP5 and myosin 1B are colocalized in E. histolytica during phagocytic cup formation. Immunoprecipitation of EhCaBP5 from total E. histolytica cellular extract also pulls out myosin 1B and this interaction was confirmed to be Ca2+ independent. Confocal imaging of E. histolytica showed that EhCaBP5 and myosin 1B are part of phagosomes. Overexpression of EhCaBP5 increases slight rate (∼20%) of phagosome formation, while suppression reduces the rate drastically (∼55%). Taken together, these experiments indicate that EhCaBP5 is likely to be the light chain of myosin 1B. Interestingly, EhCaBP5 is not present in the phagosome after its formation suggesting EhCaBP5 may be playing a regulatory role.

Structural elucidation of a dual-activity PAP phosphatase-1 from Entamoeba histolytica capable of hydrolysing both 3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate.

The enzyme 3'-phosphoadenosine 5'-phosphatase-1 (PAP phosphatase-1) is a member of the Li(+)-sensitive Mg(2+)-dependent phosphatase superfamily, or inositol monophosphatase (IMPase) superfamily, and is an important regulator of the sulfate-activation pathway in all living organisms. Inhibition of this enzyme leads to accumulation of the toxic byproduct 3'-phosphoadenosine 5'-phosphate (PAP), which could be lethal to the organism. Genomic analysis of Entamoeba histolytica suggests the presence of two isoforms of PAP phosphatase. The PAP phosphatase-1 isoform of this organism is shown to be active over wide ranges of pH and temperature. Interestingly, this enzyme is inhibited by submillimolar concentrations of Li(+), while being insensitive to Na(+). Interestingly, the enzyme showed activity towards both PAP and inositol 1,4-bisphosphate and behaved as an inositol polyphosphate 1-phosphatase. Crystal structures of this enzyme in its native form and in complex with adenosine 5'-monophosphate have been determined to 2.1 and 2.6 Šresolution, respectively. The PAP phosphatase-1 structure is divided into two domains, namely α+ß and α/ß, and the substrate and metal ions bind between them. This is a first structure of any PAP phosphatase to be determined from a human parasitic protozoan. This enzyme appears to function using a mechanism involving three-metal-ion assisted catalysis. Comparison with other structures indicates that the sensitivity to alkali-metal ions may depend on the orientation of a specific catalytic loop.

Crystal structure and mode of helicase binding of the C-terminal domain of primase from Helicobacter pylori.

To better understand the poor conservation of the helicase binding domain of primases (DnaGs) among the eubacteria, we determined the crystal structure of the Helicobacter pylori DnaG C-terminal domain (HpDnaG-CTD) at 1.78 Å. The structure has a globular subdomain connected to a helical hairpin. Structural comparison has revealed that globular subdomains, despite the variation in number of helices, have broadly similar arrangements across the species, whereas helical hairpins show different orientations. Further, to study the helicase-primase interaction in H. pylori, a complex was modeled using the HpDnaG-CTD and HpDnaB-NTD (helicase) crystal structures using the Bacillus stearothermophilus BstDnaB-BstDnaG-CTD (helicase-primase) complex structure as a template. By using this model, a nonconserved critical residue Phe534 on helicase binding interface of DnaG-CTD was identified. Mutation guided by molecular dynamics, biophysical, and biochemical studies validated our model. We further concluded that species-specific helicase-primase interactions are influenced by electrostatic surface potentials apart from the critical hydrophobic surface residues.

Machine learning-based modulation of Ca2+-binding affinity in EF-hand proteins and comparative structural insights into site-specific cooperative binding.

Ca2+-binding proteins are present in almost all living organisms and different types display different levels of binding affinities for the cation. Here, we report two new scoring schemes enabling the user to estimate and manipulate the calcium binding affinities in EF hand containing proteins. To validate this, we designed a unique EF-hand loop capable of binding calcium with high affinity by altering five residues. The N-terminal domain of Entamoeba histolytica calcium-binding protein1 (NtEhCaBP1) is used for site-directed mutagenesis to incorporate the designed loop sequence into the second EF-hand motif of this protein, referred as Nt-EhCaBP1-EF2 mutant. The binding isotherms calculated using ITC calorimetry showed that Nt-EhCaBP1-EF2 mutant site binds Ca2+ with higher affinity than Wt-Nt-EhCaBP1, by ∼600 times. The crystal structure of the mutant displayed more compact Ca2+-coordination spheres in both of its EF loops than the structure of the wildtype protein. The compact coordination sphere of EF-2 causes the bend in the helix-3, which leads to the formation of unexpected hexamer of NtEhCaBP1-EF2 mutant structure. Further dynamic correlation analysis revealed that the mutation in the second EF loop changed the entire residue network of the monomer, resulting in stronger coordination of Ca2+ even in another EF-hand loop.

Crystal structure and trimer-monomer transition of N-terminal domain of EhCaBP1 from Entamoeba histolytica.

EhCaBP1 is a well-characterized calcium binding protein from Entamoeba histolytica with four canonical EF-hand motifs. The crystal structure of EhCaBP1 reveals the trimeric organization of N-terminal domain. The solution structure obtained at pH 6.0 indicated its monomeric nature, similar to that of calmodulin. Recent domain-wise studies showed clearly that the N-terminal domain of EhCaBP1 is capable of performing most of the functions of the full-length protein. Additionally, the mode of target binding in the trimer is similar to that found in calmodulin. To study the dynamic nature of this protein and further validate the trimerization of N-terminal domain at physiological conditions, the crystal structure of N-terminal domain was determined at 2.5 A resolution. The final structure consists of EF-1 and EF-2 motifs separated by a long straight helix as seen in the full-length protein. The spectroscopic and stability studies, like far and near-ultraviolet circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, acrylamide quenching, thermal denaturation, and dynamic light scattering, provided clear evidence for a conversion from trimeric state to monomeric state. As the pH was lowered from the physiological pH, a dynamic trimer-monomer transition was observed. The trimeric state and monomeric state observed in spectroscopic studies may represent the x-ray and NMR structures of the EhCaBP1. At pH 6.0, the endogenous kinase activation function was almost lost, indicating that the monomeric state of the protein, where EF-hand motifs are far apart, is not a functional state.

Structural and functional diversity of Entamoeba histolytica calcium-binding proteins.

Entamoeba histolytica (E. histolytica) is an etiological agent of human amoebic colitis, and it causes a high level of morbidity and mortality worldwide, particularly in developing countries. Ca2+ plays a pivotal role in amoebic pathogenesis, and Ca2+-binding proteins (CaBPs) of E. histolytica appear to be a major determinant in this process. E. histolytica has 27-EF-hand containing CaBPs, suggesting that this organism has complex Ca2+ signaling cascade. E. histolytica CaBPs share (29-47%) sequence identity with ubiquitous Ca2+-binding protein calmodulin (CaM); however, they do not show any significant structural similarity, indicating lack of a typical CaM in this organism. Structurally, these CaBPs are very diverse among themselves, and perhaps such diversity allows them to recognize different cellular targets, thereby enabling them to perform a range of cellular functions. The presence of such varied signaling molecules helps parasites to invade host cells and advance in disease progression. In the past two decades, tremendous progress has been made in understanding the structure of E. histolytica CaBPs by using the X-ray or NMR method. To gain greater insight into the structural and functional diversity of these amoebic CaBPs, we analyzed and compiled all the available literature. Most of the CaBPs has about 150 amino acids with 4-EF hand or EF-hand-like sequences, similar to CaM. In a few cases, all the EF-hand motifs are not capable of binding Ca2+, suggesting them to be pseudo EF-hand motifs. The CaBPs perform diverse cellular signaling that includes cytoskeleton remodeling, phagocytosis, cell proliferation, migration of trophozoites, and GTPase activity. Overall, the structural and functional diversity of E. histolytica CaBPs compiled here may offer a basis to develop an efficient drug to counter its pathogenesis.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Unlike processive cellular motors such as myosin V, whose structure has recently been determined in a "rigor-like" conformation, myosin II from contracting muscle filaments necessarily spends most of its time detached from actin. By using squid and sea scallop sources, however, we have now obtained similar rigor-like atomic structures for muscle myosin heads (S1). The significance of the hallmark closed actin-binding cleft in these crystal structures is supported here by actin/S1-binding studies. These structures reveal how different duty ratios, and hence cellular functions, of the myosin isoforms may be accounted for, in part, on the basis of detailed differences in interdomain contacts. Moreover, the rigor-like position of switch II turns out to be unique for myosin V. The overall arrangements of subdomains in the motor are relatively conserved in each of the known contractile states, and we explore qualitatively the energetics of these states.

N-terminal residues are crucial for quaternary structure and active site conformation for the phosphoserine aminotransferase from enteric human parasite E. histolytica.

Phosphoserine aminotransferase (PSAT) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the second reversible step in the phosphoserine biosynthetic pathway producing serine. The crystal structure of E. histolytica PSAT (EhPSAT) complexed with PLP was elucidated at 3.0 Šresolution and the structures of its mutants, EhPSAT_Δ45 and EhPSAT_Δ4, at 1.8 and 2.4 Šresolution respectively. Deletion of 45 N-terminal residues (EhPSAT_Δ45) resulted in an inactive protein, the structure showed a dimeric arrangement drastically different from that of the wild-type protein, with the two monomers translated and rotated by almost 180° with respect to each other; causing a rearrangement of the active site to which PLP was unable to bind. Deletion of first N-terminal 15 (EhPSAT_Δ15) and four 11th to 14th residues (EhPSAT_Δ4) yielded up to 98% and 90% decrease in the activity respectively. Absence of aldimine linkage between PLP-Lys in the crystal structure of EhPSAT_Δ4 mutant explains for such decrease in activity and describes the importance of these N-terminal residues. Furthermore, a halide-binding site was found in close proximity to the active site. A stretch of six amino acids (146-NNTIYG-151) only conserved in the Entamoeba genus, contributes to halide binding may explain that the halide inhibition could be specific to Entamoeba.

Crystal structure of native O-acetyl-serine sulfhydrylase from Entamoeba histolytica and its complex with cysteine: structural evidence for cysteine binding and lack of interactions with serine acetyl transferase.

Cysteine plays a major role in the antioxidative defense mechanisms of the human parasite Entameoba histolytica. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine biosynthetic pathway involving two key enzymes O-acetyl-L-serine sulfhydrylase (OASS) and serine acetyl transferase (SAT). The crystal structure of native OASS from Entameoba histolytica (EhOASS) has been determined at 1.86 A resolution and in complex with its product cysteine at 2.4 A resolution. In comparison with other known OASS structures, insertion in the N-terminal region and C-terminal helix reveal critical differences, which may influence the protein-protein interactions. In spite of lacking chloride binding site at the dimeric interface, the N-terminal extension compared with other known cysteine synthases, participates in dimeric interactions in an interesting domain swapping manner, enabling it to form a stronger dimer. Sulfate is bound in the active site of the native structure, which is replaced by cysteine in the cysteine bound form causing reorientation of the small N-terminal domain and thus closure of the active site. Ligand binding constants of OAS, Cys, and Met with EhOASS are comparable with other known OASS indicating similar active site arrangement and dynamics. The cysteine complexed structure represents the snapshot of the enzyme just before releasing the final product with a closed active site. The C-terminal helix positioning in the EhOASS may effect its interactions with EhSAT and thus influencing the formation of the cysteine synthase complex in this organism.

Crystal structure of calcium binding protein-1 from Entamoeba histolytica: a novel arrangement of EF hand motifs.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Several EF-hand containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though these proteins have very high sequence similarity, they bind to different target proteins in a Ca2+ dependent manner, leading to different functional pathways (Yadava et al., Mol Biochem Parasito 1997;84:69-82; Chakrabarty et al., J Biol Chem 2004;279:12898-12908) The crystal structure of the Entamoeba histolytica calcium binding protein-1 (EhCaBP1) has been determined at 2.4 A resolution. The crystals were grown using MPD as precipitant and they belong to P6(3) space group with unit cell parameters of a = 95.25 A, b = 95.25 A, c = 64.99 A. Only two out of the four expected EF hand motifs could be modeled into the electron density map and the final model refined to R factor of 25.6% and Free_R of 28%. Unlike CaM, the first two EF hand motifs in EhCaBP1 are connected by a long helix and form a dumbbell shaped structure. Owing to domain swapping oligomerization three EhCaBP1 molecules interact in a head to tail manner to form a triangular trimer. This arrangement allows the EF-hand motif of one molecule to interact with that of an adjacent molecule to form a two EF-hand domain similar to that seen in the N-terminal domain of the NMR structure of CaBP1, calmodulin and troponin C. The oligomeric state of EhCaBP1 results in reduced flexibility between domains and may be responsible for the more limited set of targets recognized by EhCaBP1.

Crystallization and preliminary crystallographic analysis of cysteine synthase from Entamoeba histolytica.

Entamoeba histolytica, the causative agent of human amoebiasis, is essentially anaerobic, requiring a small amount of oxygen for growth. It cannot tolerate the higher concentration of oxygen present in human tissues or blood. However, during tissue invasion it is exposed to a higher level of oxygen, leading to oxygen stress. Cysteine, which is a vital thiol in E. histolytica, plays an essential role in its oxygen-defence mechanisms. The major route of cysteine biosynthesis in this parasite is the condensation of O-acetylserine with sulfide by the de novo cysteine-biosynthetic pathway, which involves cysteine synthase (EhCS) as a key enzyme. In this study, EhCS was cloned, expressed in Escherichia coli and purified by affinity and size-exclusion chromatography. The purified protein was crystallized in space group P4(1) with two molecules per asymmetric unit and a complete data set was collected to a resolution of 1.86 A. A molecular-replacement solution was obtained using the Salmonella typhimurium O-acetylserine sulfhydrylase structure as a probe and had a correlation coefficient of 37.7% and an R factor of 48.8%.

Crystal structure of scallop Myosin s1 in the pre-power stroke state to 2.6 a resolution: flexibility and function in the head.

We have extended the X-ray structure determination of the complete scallop myosin head in the pre-power stroke state to 2.6 A resolution, allowing an atomic comparison of the three major (weak actin binding) states of various myosins. We can now account for conformational differences observed in crystal structures in the so-called "pliant region" at the motor domain-lever arm junction between scallop and vertebrate smooth muscle myosins. A hinge, which may contribute to the compliance of the myosin crossbridge, has also been identified for the first time within the regulatory light-chain domain of the lever arm. Analysis of temperature factors of key joints of the motor domain, especially the SH1 helix, provides crystallographic evidence for the existence of the "internally uncoupled" state in diverse isoforms. The agreement between structural and solution studies reinforces the view that the unwinding of the SH1 helix is a part of the cross-bridge cycle in many myosins.

Response to Errors in Crystal structure of HINT from Helicobacter pylori.

A response is published to a Letter to the Editor by Maize [(2016), Acta Cryst. F72, 336-337].

Crystal structure of HINT from Helicobacter pylori.

Proteins belonging to the histidine triad (HIT) superfamily bind nucleotides and use the histidine triad motif to carry out dinucleotidyl hydrolase, nucleotidyltransferase and phosphoramidite hydrolase activities. Five different branches of this superfamily are known to exist. Defects in these proteins in humans are linked to many diseases such as ataxia, diseases of RNA metabolism and cell-cycle regulation, and various types of cancer. The histidine triad nucleotide protein (HINT) is nearly identical to proteins that have been classified as protein kinase C-interacting proteins (PKCIs), which also have the ability to bind and inhibit protein kinase C. The structure of HINT, which exists as a homodimer, is highly conserved from humans to bacteria and shares homology with the product of fragile histidine triad protein (FHit), a tumour suppressor gene of this superfamily. Here, the structure of HINT from Helicobacter pylori (HpHINT) in complex with AMP is reported at a resolution of 3 Å. The final model has R and Rfree values of 26 and 28%, respectively, with good electron density. Structural comparison with previously reported homologues and phylogenetic analysis shows H. pylori HINT to be the smallest among them, and suggests that it branched out separately during the course of evolution. Overall, this structure has contributed to a better understanding of this protein across the animal kingdom.

Structural and functional insights into the stationary-phase survival protein SurE, an important virulence factor of Brucella abortus.

The stationary-phase survival protein SurE from Brucella abortus (BaSurE) is a metal-dependent phosphatase that is essential for the survival of this bacterium in the stationary phase of its life cycle. Here, BaSurE has been biochemically characterized and its crystal structure has been determined to a resolution of 1.9 Å. BaSurE was found to be a robust enzyme, showing activity over wide ranges of temperature and pH and with various phosphoester substrates. The active biomolecule is a tetramer and each monomer was found to consist of two domains: an N-terminal domain, which forms an approximate α + ß fold, and a C-terminal domain that belongs to the α/ß class. The active site lies at the junction of these two domains and was identified by the presence of conserved negatively charged residues and a bound Mg(2+) ion. Comparisons of BaSurE with its homologues have revealed both common features and differences in this class of enzymes. The number and arrangement of some of the equivalent secondary structures, which are seen to differ between BaSurE and its homologues, are responsible for a difference in the size of the active-site area and the overall oligomeric state of this enzyme in other organisms. As it is absent in mammals, it has the potential to be a drug target.

Sequence and crystal structure determination of a basic phospholipase A2 from common krait (Bungarus caeruleus) at 2.4 A resolution: identification and characterization of its pharmacological sites.

This is the first phospholipase A2 (PLA2) structure from the family of kraits. The protein was isolated from Bungarus caeruleus (common krait) and the primary sequence was determined using cDNA approach. Three-dimensional structure of this presynaptic neurotoxic PLA2 from group I has been determined by molecular replacement method using the model of PLA2 component of beta2-bungarotoxin (Bungarus multicinctus) and refined using CNS package to a final R-factor of 20.1 % for all the data in resolution range 20.0-2.4 A. The final refined model comprises 897 protein atoms and 77 water molecules. The overall framework of krait phospholipase A2 with three long helices and two short antiparallel beta-strands is extremely similar to those observed for other group I PLA2s. However, the critical parts of PLA2 folding are concerned with its various functional loops. The conformations of these loops determine the efficiency of enzyme action and presence/absence of various pharmacological functions. In the present structure calcium-binding loop is occupied by a sodium ion with a 7-fold co-ordination. The conformation of loop 55-75 in krait PLA2 corresponds to a very high activity of the enzyme. A comparison of its sequence with multimeric PLA2s clearly shows the absence of critical residues such as Tyr3, Trp61 and Phe64, which are involved in the multimerization of PLA2 molecules. The protein shows anticoagulant and neurotoxic activities.

Crystallization and preliminary crystallographic analysis of calcium-binding protein-2 from Entamoeba histolytica and its complexes with strontium and the IQ1 motif of myosin V.

Calcium plays a pivotal role in the pathogenesis of amoebiasis, a major disease caused by Entamoeba histolytica. Two domains with four canonical EF-hand-containing calcium-binding proteins (CaBPs) have been identified from E. histolytica. Even though they have very high sequence similarity, these bind to different target proteins in a Ca2+-dependent manner, leading to different functional pathways. Calcium-binding protein-2 (EhCaBP2) crystals were grown using MPD as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 111.74, b = 68.83, c = 113.25 A, beta = 116.7 degrees. EhCaBP2 also crystallized in complex with strontium (replacing calcium) at similar conditions. The crystals belong to space group P2(1), with unit-cell parameters a = 69.18, b = 112.03, c = 93.42 A, beta = 92.8 degrees. Preliminary data for EhCaBP2 crystals in complex with an IQ motif are also reported. This complex was crystallized with MPD and ethanol as precipitating agents. These crystals belong to space group P2(1), with unit-cell parameters a = 60.5, b = 69.86, c = 86.5 A, beta = 97.9 degrees.

Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 A resolution.

The venom of the common Indian krait (Bungarus caeruleus) contains about a dozen isoforms of phospholipase A2 (PLA2), which exist in different oligomeric forms as well as in complexes with low-molecular-weight ligands. The basic objective of multimerization and complexation is either to inactivate PLA2 in the venom for long-term storage, to generate a new PLA2 function or to make a more lethal assembly. The current isoform was isolated from the venom of B. caeruleus. Dynamic light-scattering studies indicated the presence of a stable trimeric association of this PLA2. Its primary sequence was determined by cDNA cloning. The purified protein was crystallized with 2.8 M NaCl as a precipitating agent using the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 80.9, b = 80.5, c = 57.1 A, beta = 90.3 degrees. The structure was refined to a final R factor of 0.198. This is a novel trimeric PLA2 structure in which the central pore formed by the association of three molecules is filled with water molecules. The interactions across the pore take place via multiple water bridges primarily to the side chains of Arg, Lys and Thr residues. Approximately 12% of the total solvent-accessible surface area is buried in the core of the trimer. The active sites of all three molecules are located on the surface and are fully exposed to the solvent, resulting in a highly potent enzymatic unit.

Mercury induced modifications in the stereochemistry of the active site through Cys-73 in a serine protease--crystal structure of the complex of a partially modified proteinase K with mercury at 1.8 A resolution.

Proteinese K (PK) isolated from Tritirachium album Limber was crystallized with HgCl2 in excess, under microgravity conditions. The intensity data were collected at 4 degrees C to 1.8 A resolution and the final R-factor after refinement for all the reflections was 0.164. Mercury has been found at two sites with partial occupancies (0.4 and 0.6) which are at distances of 2.48 A and 2.58 A respectively from Cys-73 Sgamma. The Cys-73 in the enzyme structure is located close to the active site residue, His-69. This region is completely buried and is not accessible to the solvent. It is rather tightly packed. Therefore, the binding of mercury distorts the stereochemistry of the neighbouring residues including those belonging to the catalytic triad. As a result of this, the Ogamma of Ser-224 is displaced by 0.6 A which causes the inactivation of proteinase K by increasing the H-bond distance to 3.7 A between Ser-224 Ogamma and His-69 Nepsilon2.

Single residue mutation in active site of serine acetyltransferase isoform 3 from Entamoeba histolytica assists in partial regaining of feedback inhibition by cysteine.

The cysteine biosynthetic pathway is essential for survival of the protist pathogen Entamoeba histolytica, and functions by producing cysteine for countering oxidative attack during infection in human hosts. Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS) are involved in cysteine biosynthesis and are present in three isoforms each. While EhSAT1 and EhSAT2 are feedback inhibited by end product cysteine, EhSAT3 is nearly insensitive to such inhibition. The active site residues of EhSAT1 and of EhSAT3 are identical except for position 208, which is a histidine residue in EhSAT1 and a serine residue in EhSAT3. A combination of comparative modeling, multiple molecular dynamics simulations and free energy calculation studies showed a difference in binding energies of native EhSAT3 and of a S208H-EhSAT3 mutant for cysteine. Mutants have also been generated in vitro, replacing serine with histidine at position 208 in EhSAT3 and replacing histidine 208 with serine in EhSAT1. These mutants showed decreased affinity for substrate serine, as indicated by K(m), compared to the native enzymes. Inhibition kinetics in the presence of physiological concentrations of serine show that IC50 of EhSAT1 increases by about 18 folds from 9.59 µM for native to 169.88 µM for H208S-EhSAT1 mutant. Similar measurements with EhSAT3 confirm it to be insensitive to cysteine inhibition while its mutant (S208H-EhSAT3) shows a gain of cysteine inhibition by 36% and the IC50 of 3.5 mM. Histidine 208 appears to be one of the important residues that distinguish the serine substrate from the cysteine inhibitor.