Purification and characterization of cysteine protease of Sarcocystis fusiformis from infected Egyptian water buffaloes.

Sarcocystis spp. infects water buffaloes (Bubalus bubalis) causing sarcocystosis. In the present study, Sarcocystis fusiformis was recognized in Egyptian water buffaloes based on histological observation and molecular analysis of internal transcribed spacer 1 (ITS1), 18S ribosomal RNA (18S rRNA) and cytochrome c oxidase subunit I (COX-1) gene fragments. Chemotherapy and vaccines against Sarcocystis spp. could potentially target proteases because they may play a crucial role in the infection. Cysteine proteases are multifunctional enzymes involved in vital metabolic processes. However, the involvement of proteases in S. fusiform infection has not yet been characterized. Here, the purification and study on some biochemical properties of protease isolated from cysts of S. fusiform were carried out. Protease with a molecular weight of 100 kDa was purified. LC-MS/MS analyzed the protein sequence of purified protease and the data suggested that the enzyme might be related to the cysteine protease. The purified protease exhibited maximum activity at pH 6 and a temperature of 50 °C. The Michaelis-Menten constant (Km), the maximum velocity (Vmax), and the turnover number (Kcat) were determined. The complete inhibition effect of cysteine inhibitors indicated that the purified enzyme is a cysteine protease. The results suggested that S. fusiform proteolytic enzyme may be necessary for parasite survival in water buffaloes by digesting host tissues. Therefore, cysteine protease could be a suitable target for vaccinations.

Improving the catalytic efficiency of thermostable Geobacillus stearothermophilus xylanase XT6 by single-amino acid substitution.

Directed evolution using error-prone polymerase chain reaction was employed in the current study to enhance the catalytic efficiency of a thermostable Geobacillus stearothermophilus xylanase XT6 parent. High-throughput screening identified two variants with enhanced activity. Sequencing analysis revealed the presence of a single-amino acid substitution (P209L or V161L) in each variant. The maximum activity of mutant V161L and P209L was at 85°C and 70°C, respectively. Both mutants exhibited maximum activity at pH 7. The thermal and alkaline tolerance of mutant V161L only were markedly improved. The two mutants were more resistant to ethanol inhibition than the parent. Substrate specificity of the two mutants was shifted from beechwood xylan to birchwood xylan. The potential of the two mutants to hydrolyze rice straw and sugarcane bagasse increased. Both turnover number (kcat) and catalytic efficiency (kcat/kM) increased 12.2- and 5.7-folds for variant P209L and 13- and 6.5-folds for variant V161L, respectively, towards birchwood xylan. Based on the previously published crystal structure of extracellular G. stearothermophilus xylanase XT6, V161L and P209L mutation locate on ßα-loops. Conformational changes of the respective loops could potentiate the loop swinging, product release and consequently result in enhancement of the catalytic performance.

Revealing of a novel xylose-binding site of Geobacillus stearothermophilus xylanase by directed evolution.

Xylan saccharification is a key step in many important biotechnological applications. Xylose is the main product of xylan degradation and is a major xylanase inhibitor in a bioreactor; however, xylose-binding site of xylanase is not discovered yet. Evolving of xylose-tolerant xylanase variants will reduce the cost of xylanases in industry. Glycoside hydrolase family-10 thermostable Geobacillus stearothermophilus xylanase XT6 is non-competitively inhibited by xylose with inhibition constant ki equals to 12.2 mM. In the absence of X-ray crystallography of xylanase-xylose complex, unbiased random mutagenesis of the whole xylanase gene was done by error-prone polymerase chain reaction constructing a huge library. Screening a part of the library revealed xylose-tolerant mutants having three mutations, M116I, L131P and L133V, clustered in the N-terminus of α-helix 3. The best xylose-tolerant mutant showed higher ki and catalytic capability than that of the parent by 3.5- and 3-fold, respectively. In addition, kcat increased 4.5-fold and KM decreased 2-fold. The molecular docking of xylose into xylanase XT6 structure showed that xylose binds into a small pocket between N-terminus of α-helices 3 and 4 and close to the three mutations. Mobility of α-helices 3 and 4, which controls catalysis rate, is restricted by xylose binding and increased by these mutations.

Identification of new inhibitors for human hematopoietic prostaglandin D2 synthase among FDA-approved drugs and other compounds.

OBJECTIVE: Hematopoietic prostaglandin D2 synthase (HPGDS) is a member of the Sigma class glutathione transferases (GSTs) catalyzing the isomerization of prostaglandin H2 to prostaglandin D2, a mediator of allergy and inflammation responses. Selective inhibitors of human HPGDS are expected to be of therapeutic importance in relieving symptoms related to allergy and asthma. Hence, a collection of diverse FDA-approved compounds was screened for potential novel applications as inhibitors of HPGDS. METHODS: The catalytic activity of purified HPGDS was used for inhibition studies in vitro. RESULTS: Our inhibition studies revealed 23 compounds as effective inhibitors of HPGDS with IC50 values in the low micromolar range. Erythrosine sodium, suramin, tannic acid and sanguinarine sulfate were characterized with IC50 values of 0.2, 0.3, 0.4, and 0.6 µM, respectively. Kinetic inhibition analysis showed that erythrosine sodium is a nonlinear competitive inhibitor of HPGDS, while suramin, tannic acid and sanguinarine sulfate are linear competitive inhibitors. CONCLUSION: The results show that certain FDA-approved compounds may have pharmacological effects not previously realized that warrant further consideration in their clinical use.

In-vitro evaluation of selected Egyptian traditional herbal medicines for treatment of Alzheimer disease.

BACKGROUND: Egyptians recognized the healing power of herbs and used them in their medicinal formulations. Nowadays, "Attarin" drug shops and the public use mainly the Unani medicinal system for treatment of their health problems including improvement of memory and old age related diseases. Numerous medicinal plants have been described in old literature of Arabic traditional medicine for treatment of Alzheimer's disease (AD) (or to strengthen memory). METHODS: In this study, some of these plants were evaluated against three different preliminary bioassays related to AD to explore the possible way of their bio-interaction. Twenty three selected plants were extracted with methanol and screened in vitro against acetylcholinesterase (AChE) and cycloxygenase-1 (COX-1) enzymes. In addition, anti-oxidant activity using DPPH was determined. RESULTS: Of the tested plant extracts; Adhatoda vasica and Peganum harmala showed inhibitory effect on AChE at IC50 294 µg/ml and 68 µg/ml respectively. Moreover, A. vasica interacted reversibly with the enzyme while P. harmala showed irreversible inhibition. Ferula assafoetida (IC50 3.2 µg/ml), Syzygium aromaticum (34.9 µg/ml) and Zingiber officinalis (33.6 µg/ml) showed activity against COX-1 enzyme. Potent radical scavenging activity was demonstrated by three plant extracts Terminalia chebula (EC50 2.2 µg/ml), T. arjuna (3.1 µg/ml) and Emblica officinalis (6.3 µg/ml). CONCLUSION: Interestingly, differential results have been obtained which indicate the variability of the mode of actions for the selected plants. Additionally, the reversible interaction of A. vasica against AChE and the potent activity of F. assafoetida against COX-1 make them effective, new and promising agents for treatment of AD in the future, either as total extracts or their single bioactive constituents.

FDA-approved drugs and other compounds tested as inhibitors of human glutathione transferase P1-1.

OBJECTIVE: Glutathione transferase P1-1 (GST P1-1) is often overexpressed in tumor cells and is regarded as a contributor to their drug resistance. Inhibitors of GST P1-1 are expected to counteract drug resistance and may therefore serve as adjuvants in the chemotherapy of cancer by increasing the efficacy of cytostatic drugs. Finding useful inhibitors among compounds used for other indications would be a shortcut to clinical applications and a search for GST P1-1 inhibitors among approved drugs and other compounds was therefore conducted. METHODS: We tested 1040 FDA-approved compounds as inhibitors of the catalytic activity of purified human GST P1-1 in vitro. RESULTS: We identified chlorophyllide, merbromine, hexachlorophene, and ethacrynic acid as the most effective GST P1-1 inhibitors with IC50 values in the low micromolar range. For comparison, these compounds were even more potent in the inhibition of human GST A3-3, an enzyme implicated in steroid hormone biosynthesis. In distinction from the other inhibitors, which showed conventional inhibition patterns, the competitive inhibitor ethacrynic acid elicited strong kinetic cooperativity in the glutathione saturation of GST P1-1. Apparently, ethacrynic acid serves as an allosteric inhibitor of the enzyme. CONCLUSION AND PRACTICAL IMPLICATIONS: In their own right, the compounds investigated are less potent than desired for adjuvants in cancer chemotherapy, but the structures of the most potent inhibitors could serve as leads for the synthesis of more efficient adjuvants.

Hidden allostery in human glutathione transferase p1-1 unveiled by unnatural amino acid substitutions and inhibition studies.

Conventional steady-state kinetic studies of the dimeric human glutathione transferase (GST) P1-1 do not reveal obvious deviations from Michaelis-Menten behavior. By contrast, engineering of the key residue Y50 of the lock-and-key motif in the subunit interface reveals allosteric properties of the enzyme. The low-activity mutant Y50C, characterized by 150-fold decreased kcat and 300-fold increased KM(GSH) values, displays an apparent Hill coefficient of 0.82±0.22. Chemical alkylation of the sulfhydryl group of Y50C by unnatural n-butyl or n-pentyl substitutions enhances the catalytic efficiency kcat/KM(GSH) to near the wild-type value but still yields Hill coefficients of 0.61±0.08 and 0.86±0.1, respectively. Thus, allosteric kinetic behavior is not dependent on low activity of the enzyme. On the other hand, S-cyclobutylmethyl-substituted Y50C, which also displays high catalytic efficiency, has a Hill coefficient of 0.99±0.11, showing that subtle differences in structure at the subunit interface influence the complex kinetic behavior. Furthermore, inhibition studies of native GST P1-1 using ethacrynic acid demonstrate that a ligand bound noncovalently to the wild-type enzyme also can elicit allosteric kinetic behavior. Thus, we conclude that the GST P1-1 structure has intrinsic allostery that becomes overt under some, but not all, ambient conditions.

Modulating catalytic activity by unnatural amino acid residues in a GSH-binding loop of GST P1-1.

The loop following helix alpha2 in glutathione transferase P1-1 has two conserved residues, Cys48 and Tyr50, important for glutathione (GSH) binding and catalytic activity. Chemical modification of Cys48 thwarts the catalytic activity of the enzyme, and mutation of Tyr50 generally decreases the k(cat) value and the affinity for GSH in a differential manner. Cys48 and Tyr50 were targeted by site-specific mutations and chemical modifications in order to investigate how the alpha2 loop modulates GSH binding and catalysis. Mutation of Cys48 into Ala increased K(M)(GSH) 24-fold and decreased the binding energy of GSH by 1.5 kcal/mol. Furthermore, the protein stability against thermal inactivation and chemical denaturation decreased. The crystal structure of the Cys-free variant was determined, and its similarity to the wild-type structure suggests that the mutation of Cys48 increases the flexibility of the alpha2 loop rather than dislocating the GSH-interacting residues. On the other hand, replacement of Tyr50 with Cys, producing mutant Y50C, increased the Gibbs free energy of the catalyzed reaction by 4.8 kcal/mol, lowered the affinity for S-hexyl glutathione by 2.2 kcal/mol, and decreased the thermal stability. The targeted alkylation of Cys50 in Y50C increased the affinity for GSH and protein stability. Characterization of the most active alkylated variants, S-n-butyl-, S-n-pentyl-, and S-cyclobutylmethyl-Y50C, indicated that the affinity for GSH is restored by stabilizing the alpha2 loop through positioning of the key residue into the lock structure of the neighboring subunit. In addition, k(cat) can be further modulated by varying the structure of the key residue side chain, which impinges on the rate-limiting step of catalysis.

Replacement surgery with unnatural amino acids in the lock-and-key joint of glutathione transferase subunits.

Proteins contain amino acid residues essential to structure and function. Ribosomal protein synthesis is typically limited to the 20 amino acids of the genetic code, but posttranslational chemical modifications can greatly expand the diversity of side chain functionalities. In this investigation, a natural aromatic residue in the lock-and-key joint at the subunit interface of the dimeric glutathione transferase P1-1 was replaced by an S-alkylcysteine residue to give a functional enzyme. Introduction of Cys in the key position inactivates the enzyme, but subsequent alkylation of this residue enhances the catalytic efficiency up to 27,000-fold. Combinatorial modification of Cys by a mixture of reagents facilitated identification of an n-butyl group as the most efficient activator. Alkylation also enhanced binding affinity for active-site ligands and stabilized the enzyme against chemical denaturation and thermal inactivation.

Functional role of the lock and key motif at the subunit interface of glutathione transferase p1-1.

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins. Each subunit has an active site, but there is no evidence for the existence of catalytically active monomers. The lock and key motif is responsible for a highly conserved hydrophobic interaction in the subunit interface of pi, mu, and alpha class glutathione transferases. The key residue, which is either Phe or Tyr (Tyr(50) in human GSTP1-1) in one subunit, is wedged into a hydrophobic pocket of the other subunit. To study how an essentially inactive subunit influences the activity of the neighboring subunit, we have generated the heterodimer composed of subunits from the fully active human wild-type GSTP1-1 and the nearly inactive mutant Y50A obtained by mutation of the key residue Tyr(50) to Ala. Although the key residue is located far from the catalytic center, the k(cat) value of mutant Y50A decreased about 1300-fold in comparison with the wild-type enzyme. The decrease of the k(cat) value of the heterodimer by about 27-fold rather than the expected 2-fold in comparison with the wild-type enzyme indicates that the two active sites of the dimeric enzyme work synergistically. Further evidence for cooperativity was found in the nonhyperbolic GSH saturation curves. A network of hydrogen-bonded water molecules, found in crystal structures of GSTP1-1, connects the two active sites and the main chain carbonyl group of Tyr(50), thereby offering a mechanism for communication between the two active sites. It is concluded that a subunit becomes catalytically competent by positioning the key residue of one subunit into the lock pocket of the other subunit, thereby stabilizing the loop following the helix alpha2, which interacts directly with GSH.