Molecular Prediction and Correlation of the Structure and Function of Universal Stress Protein A (UspA) from Salmonella Typhimurium.

Salmonella Typhimurium (ST) is a zoonotic pathogen that can cause gastroenteritis in humans when they consume contaminated food or water. When exposed to various stressors, both from living organisms (biotic) and the environment (abiotic), Salmonella Typhimurium produces Universal Stress Proteins (USPs). These proteins are gaining recognition for their crucial role in bacterial stress resistance and the ability to enter a prolonged state of growth arrest. Additionally, USPs exhibit diverse structures due to the fusion of the USP domain with different catalytic motifs, enabling them to participate in various reactions and cellular activities during stressful conditions. In this particular study, researchers cloned and analyzed the uspA gene obtained from poultry-derived strains of Salmonella Typhimurium. The gene comprises 435 base pairs, encoding a USP family protein consisting of 144 amino acids. Phylogenetic analysis demonstrated a close relationship between the uspA genes of Salmonella Typhimurium and those found in other bacterial species. We used molecular dynamics simulations and 3D structure prediction to ensure that the USPA protein was stable. Furthermore, we also carried out motif search and network analysis of protein-protein interactions. The findings from this study offer valuable insights for the development of inhibitors targeted against Salmonella Typhimurium.

Recurrence of Varicose Vein after Endovenous Laser Therapy in a Tertiary Care Center: A Descriptive Cross-sectional Study.

INTRODUCTION: Varicosity is the common problem of various etiology having simple limb aching to worst complications like oedema, ulcer, and skin changes. Minimal invasive endovenous laser therapy is a noble procedure. The aim of the study is to find out the recurrence of the varicose vein after laser therapy in a tertiary care center. METHODS: This descriptive cross-sectional study was done in 38 patients with varicosity of the lower limb in a tertiary care hospital, from January 2019 to June 2019 after taking ethical clearance from Institutional Review Committee. Convenience sampling was done. Data was collected and entry was done in Statistical Package for the Social Science software version 22, point estimate at 90% Confidence Interval was calculated along with frequency and proportion for binary data. RESULTS: We recorded 38 patients with ablated limb out of which none of the ablated veins showed recanalization in six months follow up. Twenty two (58%) patients were male and 16 (42%) patients were female with a mean age of 40.26 years. Major bulk, 23 (60.5%) resumed activity in second postoperative day and only 1 (2.6%) patient waited for 5 days for normal activity with mean of 2.58 days postoperatively. Sixteen (42.1%) patients developed erythema or ecchymosis, 12 (31.6%) patients had induration along the long saphenous vein course, 7 (18.4%) patients had paresthesia, 2 (5.3%) patients had limb swelling and 1 (2.6%) patient had skin burn. CONCLUSIONS: Endovenous laser ablation has very low rate of recurrence of varicosity and has minor complications.

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations.

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser-His-Asp triad, proposed to serve a general acid-base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 µmol·kg-1 in brain to 1.4 µmol·kg-1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 µm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.

Prion protein transcription is auto-regulated through dynamic interactions with G-quadruplex motifs in its own promoter.

Cellular prion protein (PrP) misfolds into an aberrant and infectious scrapie form (PrPSc) that lead to fatal transmissible spongiform encephalopathies (TSEs). Association of prions with G-quadruplex (GQ) forming nucleic acid motifs has been reported, but implications of these interactions remain elusive. Herein, we show that the promoter region of the human prion gene (PRNP) contains two putative GQ motifs (Q1 and Q2) that assume stable, hybrid, intra-molecular quadruplex structures and bind with high affinity to PrP. Here, we investigate the ability of PrP to bind to the quadruplexes in its own promoter. We used a battery of techniques including SPR, NMR, CD, MD simulations and cell culture-based reporter assays. Our results show that PrP auto-regulates its expression by binding and resolving the GQs present in its own promoter. Furthermore, we map this resolvase-like activity to the N-terminal region (residues 23-89) of PrP. Our findings highlight a positive transcriptional-translational feedback regulation of the PRNP gene by PrP through dynamic unwinding of GQs in its promoter. Taken together, our results shed light on a yet unknown mechanism of regulation of the PRNP gene. This work provides the necessary framework for a plethora of studies on understanding the regulation of PrP levels and its implications in prion pathogenesis.

Genome-Wide Analysis of HSP70 Family Protein in Vigna radiata and Coexpression Analysis Under Abiotic and Biotic Stress.

Heat shock protein 70 (Hsp70), a 70-kDa protein, also known as a molecular chaperone, is highly conserved. It plays a major role in cellular functions such as protein folding, regulation of protein degradation, translocation of proteins across membranes, receptor signaling, and protein assembly or disassembly. Vigna radiata is an important legume crop with available whole-genome sequence, but no such study on the HSP70 family is reported. A total of 32 V. radiate HSP70s (Vr-HSP70s) were identified and described. They are phylogenetically clustered into four subgroups. Vr-HSP70s show variations in intron/exon organization. This indicates that introns may play an essential role in gene regulating. The coexpression analysis of Vr-HSP70s revealed that these genes were involved in both abiotic and biotic stresses. Three cytoplasmic hub genes namely Vr-HSP70-C-14, Vr-HSP70-C-29, and Vr-HSP70-C-30 were found common in both stresses. Our findings provide directions for future studies to dissect functional analysis of Vr-HSP70s in response to abiotic and biotic stresses.

VigSatDB: genome-wide microsatellite DNA marker database of three species of Vigna for germplasm characterization and improvement.

Genus Vigna represented by more than 100 species is a source of nutritious edible seeds and sprouts that are rich sources of protein and dietary supplements. It is further valuable because of therapeutic attributes due to its antioxidant and anti-diabetic properties. A highly diverse and an extremely ecological niche of different species can be valuable genomic resources for productivity enhancement. It is one of the most underutilized crops for food security and animal feeds. In spite of huge species diversity, only three species of Vigna have been sequenced; thus, there is a need for molecular markers for the remaining species. Computational approach of microsatellite marker discovery along with evaluation of polymorphism utilizing available genomic data of different genotypes can be a quick and an economical approach for genomic resource development. Cross-species transferability by e-PCR over available genomes can further prioritize the potential SSR markers, which could be used for genetic diversity and population differentiation of the remaining species saving cost and time. We present VigSatDB-the world's first comprehensive microsatellite database of genus Vigna, containing >875 K putative microsatellite markers with 772 354 simple and 103 865 compound markers mined from six genome assemblies of three Vigna species, namely, Vigna radiata (Mung bean), Vigna angularis (Adzuki bean) and Vigna unguiculata (Cowpea). It also contains 1976 validated published markers. Markers can be selected on the basis of chromosomes/location specificity, and primers can be generated using Primer3core tool integrated at backend. Efficacy of VigSatDB for microsatellite loci genotyping has been evaluated by 15 markers over a panel of 10 diverse genotype of V. radiata. Our web genomic resources can be used in diversity analysis, population and varietal differentiation, discovery of quantitative trait loci/genes, marker-assisted varietal improvement in endeavor of Vigna crop productivity and management.

Pattern of Pediatric Oncology Cases in the Western Region of Nepal

Childhood cancers form a rare and heterogeneous group which fortunately have a higher cure rate than adult cancers. A few non-profit organizations in Nepal have extended support to help patients suffering from cancer, but their main focus has been on adults. The objective of this study was to establish the pattern of childhood cancers in the Western region of Nepal. We reviewed cases receiving external radiotherapy with both palliative and curative intent between 28th September 2010 and 30th September 2015 at the Department of Radiotherapy and Oncology, Manipal Teaching Hospital affiliated with Manipal College of Medical Sciences, Pokhara, Nepal. Of the total of 1217 cases, 2.71% involved children. The gender distribution showed a male preponderance (M:F= 1.35:1). The patients' mean age was 11.4 years (range 2 - 14 years). Considering the caste, Brahmins and Gurungs constituted 33.0% and 21.2%, respectively, of children with cancer.

Utility of Acute Normovolemic Hemodilution in Major Surgeries in Rural Area: A Prospective Comparative Study from North India.

INTRODUCTION: Easy availability of autologous blood is difficult in rural areas. Acute normovolemic hemodilution (ANH) has been found to be an effective alternative in major surgeries where we are expecting major blood loss. PATIENTS AND METHODS: A prospective comparative study was designed to evaluate the utility of ANH patients (patients receiving autologous blood) during major operations done at MRA Medical College Ambedkar Nagar, Uttar Pradesh, India. during from September 2015 to September 2016. A total of 60 patients undergoing major surgeries were randomly assigned into two groups of thirty patients' each. Group I received homologous blood intraoperative only when required. In Group II ANH was initiated to a target hematocrit of 30% after induction of anesthesia. Various parameters such as demographic, biochemical, and hemodynamic were compared. RESULTS: The mean value of blood withdrawn in ANH group was 650.5 ± 228 ml and it was replaced with an equal volume of 6% hydroethyl starch. There was no statistically significant variation in mean hemocrits levels in both the groups at various stages of the study. Hematocrits decreased significantly in both the groups at various stages as compared to preoperative values. The heart rate and mean blood pressure were almost similar and without statistically significant differences in both groups. Surgical blood loss in Group I was 895.29 ± 568.30 ml as compared to 765 ± 506 ml in Group II. The difference was statistically insignificant (P ≥ 0.05). The mean volume of homologous blood transfused in Group I was 850.71 ± 318.29 ml, as compared to nil in Group II which was statistically significant (P < 0.05). CONCLUSION: It concludes that ANH up to a target hematocrit of 30% is safe and effective in reducing the need for homologous blood in various major surgeries in institutes in rural areas.

Development of transcriptome based web genomic resources of yellow mosaic disease in Vigna mungo.

Vigna mungo (Urdbean) is cultivated in the tropical and sub-tropical continental region of Asia. It is not only important source of dietary protein and nutritional elements, but also of immense value to human health due to medicinal properties. Yellow mosaic disease caused by Mungbean Yellow Mosaic India Virus is known to incur huge loss to crop, adversely affecting crop yield. Contrasting genotypes are ideal source for knowledge discovery of plant defence mechanism and associated candidate genes for varietal improvement. Whole genome sequence of this crop is yet to be completed. Moreover, genomic resources are also not freely accessible, thus available transcriptome data can be of immense use. V. mungo Transcriptome database, accessible at http://webtom.cabgrid.res.in/vmtdb/ has been developed using available data of two contrasting varieties viz., cv. VM84 (resistant) and cv. T9 (susceptible). De novo assembly was carried out using Trinity and CAP3. Out of total 240,945 unigenes, 165,894 (68.8%) showed similarity with known genes against NR database, and remaining 31.2% were found to be novel. We found 22,101 differentially expressed genes in all datasets, 44,335 putative genic SSR markers, 4105 SNPs and Indels, 64,964 transcriptional factor, 546 mature miRNA target prediction in 703 differentially expressed unigenes and 137 pathways. MAPK, salicylic acid-binding protein 2-like, pathogenesis-related protein and NBS-LRR domain were found which may play an important role in defence against pathogens. This is the first web genomic resource of V. mungo for future genome annotation as well as ready to use markers for future variety improvement program.

Improved tag-switch method reveals that thioredoxin acts as depersulfidase and controls the intracellular levels of protein persulfidation.

Hydrogen sulfide (H2S) has emerged as a signalling molecule capable of regulating several important physiological functions such as blood pressure, neurotransmission and inflammation. The mechanisms behind these effects are still largely elusive and oxidative posttranslational modification of cysteine residues (protein persulfidation or S-sulfhydration) has been proposed as the main pathway for H2S-induced biological and pharmacological effects. As a signalling mechanism, persulfidation has to be controlled. Using an improved tag-switch assay for persulfide detection we show here that protein persulfide levels are controlled by the thioredoxin system. Recombinant thioredoxin showed an almost 10-fold higher reactivity towards cysteine persulfide than towards cystine and readily cleaved protein persulfides as well. This reaction resulted in H2S release suggesting that thioredoxin could be an important regulator of H2S levels from persulfide pools. Inhibition of the thioredoxin system caused an increase in intracellular persulfides, highlighting thioredoxin as a major protein depersulfidase that controls H2S signalling. Finally, using plasma from HIV-1 patients that have higher circulatory levels of thioredoxin, we could prove depersulfidase role in vivo.

Prevalence and Association of Developmental Defects of Enamel with, Dental- Caries and Nutritional Status in Pre-School Children, Lucknow.

BACKGROUND: Developmental Defects of Enamel in the primary dentition may be associated and predictors of dental caries and nutritional status. The aim of the present study was to assess the Prevalence of Developmental Defects of Enamel and its Association with, Dental-Caries and Nutritional Status in Pre-School Children of Lucknow, India. MATERIALS AND METHODS: Multistage Sampling was done. A total of 302 pre-school (Rural and Urban) children were examined. Type III examination was conducted with WHO Probe. Developmental Enamel Defects (DED) and Dental Caries were assessed using WHO (1997) Proforma. RESULTS: The prevalence of DED of any type was 39.9% with that of demarcated opacities being the highest, followed by hypoplasia. The most frequently affected teeth were maxillary anterior teeth, while the least affected teeth were mandibular incisors. The mean dmft was 3.5. A positive association between DED and caries was observed. Association between Dental Caries & BMI was non-significant whereas Pearson correlation showed a negative correlation between the two. CONCLUSION: The prevalence of enamel defects and caries was high, as the enamel defects were strongly associated with caries.

Haemato-biochemical and oxidative status of buffaloes naturally infected with Trypanosoma evansi.

Blood samples were collected from 05 clinically healthy and 10 adult female water buffaloes naturally infected with Trypanosoma evansi. Confirmation of disease free and infected status of buffaloes was made on clinical signs, observation of T. evansi parasites in the blood smear and duplex PCR based assay. Blood samples were evaluated for levels of haemoglobin (Hb), packed cell volume (PCV), differential leucocytes count (DLC), lipid peroxidation (LPO), calcium, phosphorous, magnesium sodium and potassium and activities of superoxide dismutase (SOD), catalase (CAT), aspartate transaminase (AST), lactate dehydogenase (LDH) and alkaline phosphatase (ALP). The results of the study revealed substantial decrease in levels of Hb, PCV and increase in LPO, SOD, CAT and AST in infected animals compared to healthy animals. However other haematological and biochemical indices did not show significant variations in infected and healthy buffaloes. The enhanced erythrocytic oxidation and reduction of hematological indices, suggests that the enhanced oxidation of the erythrocytes may be a contributory factor in erythrocytic destruction and progression of the anaemia in T. evansi infection in water buffaloes.

S-glutathionylation enhances human cystathionine ß-synthase activity under oxidative stress conditions.

AIMS: Cystathionine ß-synthase (CBS) catalyzes the first and rate-limiting step in the two-step trans-sulfuration pathway that converts homocysteine to cysteine. It is also one of three major enzymes responsible for the biogenesis of H2S, a signaling molecule. We have previously demonstrated that CBS is activated in cells challenged by oxidative stress, but the underlying molecular mechanism of this regulation has remained unclear. RESULTS: Here, we demonstrate that S-glutathionylation of CBS enhances its activity ∼2-fold in vitro. Loss of this post-translational modification in the presence of dithiothreitol results in reversal to basal activity. Cys346 was identified as the site for S-glutathionylation by a combination of mass spectrometric, mutagenesis, and activity analyses. To test the physiological relevance of S-glutathionylation-dependent regulation of CBS, HEK293 cells were oxidatively challenged with peroxide, which is known to enhance the trans-sulfuration flux. Under these conditions, CBS glutathionylation levels increased and were correlated with a ∼3-fold increase in CBS activity. INNOVATION: Collectively, our results reveal a novel post-translational modification of CBS, that is, glutathionylation, which functions as an allosteric activator under oxidative stress conditions permitting enhanced synthesis of both cysteine and H2S. CONCLUSIONS: Our study elucidates a molecular mechanism for increased cysteine and therefore glutathione, synthesis via glutathionylation of CBS. They also demonstrate the potential for increased H2S production under oxidative stress conditions, particularly in tissues where CBS is a major source of H2S.

Organization of the human mitochondrial hydrogen sulfide oxidation pathway.

Sulfide oxidation is expected to play an important role in cellular switching between low steady-state intracellular hydrogen sulfide levels and the higher concentrations where the physiological effects are elicited. Yet despite its significance, fundamental questions regarding how the sulfide oxidation pathway is wired remain unanswered, and competing proposals exist that diverge at the very first step catalyzed by sulfide quinone oxidoreductase (SQR). We demonstrate that, in addition to sulfite, glutathione functions as a persulfide acceptor for human SQR and that rhodanese preferentially synthesizes rather than utilizes thiosulfate. The kinetic behavior of these enzymes provides compelling evidence for the flow of sulfide via SQR to glutathione persulfide, which is then partitioned to thiosulfate or sulfite. Kinetic simulations at physiologically relevant metabolite concentrations provide additional support for the organizational logic of the sulfide oxidation pathway in which glutathione persulfide is the first intermediate formed.

Structure and kinetic analysis of H2S production by human mercaptopyruvate sulfurtransferase.

Mercaptopyruvate sulfurtransferase (MST) is a source of endogenous H2S, a gaseous signaling molecule implicated in a wide range of physiological processes. The contribution of MST versus the other two H2S generators, cystathionine ß-synthase and γ-cystathionase, has been difficult to evaluate because many studies on MST have been conducted at high pH and have used varied reaction conditions. In this study, we have expressed, purified, and crystallized human MST in the presence of the substrate 3-mercaptopyruvate (3-MP). The kinetics of H2S production by MST from 3-MP was studied at pH 7.4 in the presence of various physiological persulfide acceptors: cysteine, dihydrolipoic acid, glutathione, homocysteine, and thioredoxin, and in the presence of cyanide. The crystal structure of MST reveals a mixture of the product complex containing pyruvate and an active site cysteine persulfide (Cys(248)-SSH) and a nonproductive intermediate in which 3-MP is covalently linked via a disulfide bond to an active site cysteine. The crystal structure analysis allows us to propose a detailed mechanism for MST in which an Asp-His-Ser catalytic triad is positioned to activate the nucleophilic cysteine residue and participate in general acid-base chemistry, whereas our kinetic analysis indicates that thioredoxin is likely to be the major physiological persulfide acceptor for MST.

Molecular modeling, dynamics studies and virtual screening of Fructose 1, 6 biphosphate aldolase-II in community acquired- methicillin resistant Staphylococcus aureus (CA-MRSA).

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) has recently emerged as a nosocomial pathogen to the community which commonly causes skin and soft-tissue infections (SSTIs). This strain (MW2) has now become resistant to the most of the beta-lactam antibiotics; therefore it is the urgent need to identify the novel drug targets. Recently fructose 1,6 biphosphate aldolase-II (FBA) has been identified as potential drug target in CA-MRSA. The FBA catalyses the retro-ketolic cleavage of fructose-1,6-bisphosphate (FBP) to yield dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P) in glycolytic pathway. In the present research work the 3D structure of FBA was predicted using the homology modeling method followed by validation. The molecular dynamics simulation (MDS) of the predicted model was carried out using the 2000 ps time scale and 1000000 steps. The MDS results suggest that the modeled structure is stable. The predicted model of FBA was used for virtual screening against the NCI diversity subset-II ligand databases which contain 1364 compounds. Based on the docking energy scores, it was found that top four ligands i.e. ZINC01690699, ZINC13154304, ZINC29590257 and ZINC29590259 were having lower energy scores which reveal higher binding affinity towards the active site of FBA. These ligands might act as potent inhibitors for the FBA so that the menace of antimicrobial resistance in CA-MRSA can be conquered. However, pharmacological studies are required to confirm the inhibitory activity of these ligands against the FBA in CA-MRSA.

Detection of reaction intermediates during human cystathionine ß-synthase-monitored turnover and H2S production.

Human cystathionine ß-synthase (CBS), a novel heme-containing pyridoxal 5'-phosphate enzyme, catalyzes the condensation of homocysteine and serine or cysteine to produce cystathionine and H(2)O or H(2)S, respectively. The presence of heme in CBS has limited spectrophotometric characterization of reaction intermediates by masking the absorption of the pyridoxal 5'-phosphate cofactor. In this study, we employed difference stopped-flow spectroscopy to characterize reaction intermediates formed under catalytic turnover conditions. The reactions of L-serine and L-cysteine with CBS resulted in the formation of a common aminoacrylate intermediate (k(obs) = 0.96 ± 0.02 and 0.38 ± 0.01 mM(-1) s(-1), respectively, at 24 °C) with concomitant loss of H(2)O and H(2)S and without detectable accumulation of the external aldimine or other intermediates. Homocysteine reacted with the aminoacrylate intermediate with k(obs) = 40.6 ± 3.8 s(-1) and re-formed the internal aldimine. In the reverse direction, CBS reacted with cystathionine, forming the aminoacrylate intermediate with k(obs) = 0.38 ± 0.01 mM(-1) s(-1). This study provides the first insights into the pre-steady-state kinetic mechanism of human CBS and indicates that the reaction is likely to be limited by a conformational change leading to product release.

Potential therapeutic drug target identification in Community Acquired-Methicillin Resistant Staphylococcus aureus (CA-MRSA) using computational analysis.

The emergence of multidrug-resistant strain of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strain has highlighted the urgent need for the alternative and effective therapeutic approach to combat the menace of this nosocomial pathogen. In the present work novel potential therapeutic drug targets have been identified through the metabolic pathways analysis. All the gene products involved in different metabolic pathways of CA-MRSA in KEGG database were searched against the proteome of Homo sapiens using the BLASTp program and the threshold of E-value was set to as 0.001. After database searching, 152 putative targets were identified. Among all 152 putative targets, 39 genes encoding for putative targets were identified as the essential genes from the DEG database which are indispensable for the survival of CA-MRSA. After extensive literature review, 7 targets were identified as potential therapeutic drug target. These targets are Fructose-bisphosphate aldolase, Phosphoglyceromutase, Purine nucleoside phosphorylase, Uridylate kinase, Tryptophan synthase subunit beta, Acetate kinase and UDP-N-acetylglucosamine 1-carboxyvinyltransferase. Except Uridylate kinase all the identified targets were involved in more than one metabolic pathways of CA-MRSA which underlines the importance of drug targets. These potential therapeutic drug targets can be exploited for the discovery of novel inhibitors for CA-MRSA using the structure based drug design (SBDD) strategy.

Allosteric communication between the pyridoxal 5'-phosphate (PLP) and heme sites in the H2S generator human cystathionine ß-synthase.

Human cystathionine ß-synthase (CBS) is a unique pyridoxal 5'-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ~2- to ~500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H(2)S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H(2)S-generating reactions catalyzed by CBS.

Insight into trichomonas vaginalis genome evolution through metabolic pathways comparison.

Trichomonas vaginalis causes the trichomoniasis, in women and urethritis and prostate cancer in men. Its genome draft published by TIGR in 2007 presents many unusual genomic and biochemical features like, exceptionally large genome size, the presence of hydrogenosome, gene duplication, lateral gene transfer mechanism and the presence of miRNA. To understand some of genomic features we have performed a comparative analysis of metabolic pathways of the T. vaginalis with other 22 significant common organisms. Enzymes from the biochemical pathways of T. vaginalis and other selected organisms were retrieved from the KEGG metabolic pathway database. The metabolic pathways of T. vaginalis common in other selected organisms were identified. Total 101 enzymes present in different metabolic pathways of T. vaginalis were found to be orthologous by using BLASTP program against the selected organisms. Except two enzymes all identified orthologous enzymes were also identified as paralogous enzymes. Seventy-five of identified enzymes were also identified as essential for the survival of T. vaginalis, while 26 as non-essential. The identified essential enzymes also represent as good candidate for novel drug targets. Interestingly, some of the identified orthologous and paralogous enzymes were found playing significant role in the key metabolic activities while others were found playing active role in the process of pathogenesis. The N-acetylneuraminate lyase was analyzed as the candidate of lateral genes transfer. These findings clearly suggest the active participation of lateral gene transfer and gene duplication during evolution of T. vaginalis from the enteric to the pathogenic urogenital environment.