Structural insights of novel mutational frames in Bromodomain Containing-2 gene (BRD2) in juvenile myoclonic epilepsy: bed, bench, and laptop profiles.

PURPOSE: Juvenile myoclonic epilepsy (JME) is an adolescent onset type of idiopathic generalized epilepsies. Bromodomain containing protein-2 gene (BRD2), a transcriptional regulatory protein, has a susceptible role in the expression of JME. Considering the polymorphic variations observed in exon 3 of the BRD2 gene, we evaluated the molecular interactions with anti-seizure medication in individuals diagnosed with JME. METHODS: The genomic DNA was extracted from 5 mL of peripheral venous blood of JME participants (n = 55) and healthy control subjects (n = 55). Detailed anti-seizure medication and outcomes were noted during the study period. Identified novel mutations at nucleotide and protein sequences, compared by multiple sequence alignment. Wild-type (WT) and mutated-type (MT) structures were investigated for molecular docking and interactions with anti-seizure drugs. RESULTS: A common variant at c.1707G>A was found among 23 participants, while a single variant at c.1663ins C was found in one participant. The deletion positions were observed at c.1890delA, c.1892A>T, c.1895A>T, c.1896G>T, c.1897T>C, c.1898T>C, c.1899C>T, c.1900G>T, c.1901C>T and c.1902A>T exhibiting stop codon after p.111Pro>stop; these variants resulted in a truncated protein. In silico analysis was conducted to validate changes; docking analysis showed that novel variant has a significant role in the interactions with anti-seizure drugs. SIGNIFICANCE: Besides clinical and genetic outcomes, ∼5.45% unique genetical variations were observed in the participants. Significant mimicked at the binding site position (92-111) of human BRD2 ranges ∼8.2%, ∼16.4%, and ∼10.6%. Further, research is needed to identify the importance of polymorphism alterations at the binding site and their molecular interactions with anti-seizure drugs, which might be confirmed in a diverse population with JME.

Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

BACKGROUND: When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. METHODS: Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. RESULTS: Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. CONCLUSION: The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

Mutations in exons 3 and 7 resulting in truncated expression of human ATP6V1B1 gene showing structural variations contributing to poor substrate binding-causative reason for distal renal tubular acidosis with sensorineural deafness.

Distal renal tubular acidosis (dRTA) is an autosomal recessive syndrome results defect in either proximal tubule bicarbonate reabsorption or in distal tubule H(+) secretion and is characterized by severe hyperchloraemic metabolic acidosis in childhood. dRTA is associated with functional variations in the ATP6V1B1 gene encoding ß1 subunit of H(+)-ATPase, key membrane transporters for net acid excretion of α-intercalated cells of medullary collecting ducts. In the present study, a 13-year-old male patient suffering with nephropathy and sensorineural deafness was reported in the Department of Nephrology. We predicted improper functioning of ATP6V1B1 gene could be the reason for diseased condition. Therefore, exons 3, 4, and 7 contributing active site of ATP6V1B1 gene was amplified and sequenced (Accession numbers: KF571726, KM222653). The obtained sequences were BLAST searched against the wild type ATP6V1B1 gene which showed novel mutations c.307 A > G, c.308 C > A, c.310 C > G, c.704 T > C, c.705 G > T, c.709 A > G, c.710 A > G, c.714 G > A, c.716 C > A, c.717delC, c.722 C > G, c.728insG, c.741insT, c.753G > C. These mutations resulted in the expression of truncated protein terminating at Lys 209. The mutated ATP6V1B1structure superimposed with wild type showed extensive variations with RMSD 1.336 Å and could not bind to substrate ADP leading to non-functional ATPase. These results conclusively explain these mutations in ATP6V1B1 gene resulted in structural changes causing accumulation of H(+) ions contributing to dRTA with sensorineural deafness.

In silico designing and molecular docking of a potent analog against Staphylococcus aureus porphobilinogen synthase.

BACKGROUND: The emergence of multidrug-resistant strains of Staphylococcus aureus, there is an urgent need for the development of new antimicrobials which are narrow and pathogen specific. AIM: In this context, the present study is aimed to have a control on the staphylococcal infections by targeting the unique and essential enzyme; porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of δ-aminolevulinic acid, an essential step in the tetrapyrrole biosynthesis. Hence developing therapeutics targeting PBGS will be the promising choice to control and manage the staphylococcal infections. 4,5-dioxovalerate (DV) is known to inhibit PBGS. MATERIALS AND METHODS: In view of this, in this study, novel dioxovalerate derivatives (DVDs) molecules were designed so as to inhibit PBGS, a potential target of S. aureus and their inhibitory activity was predicted using molecular docking studies by molecular operating environment. The 3D model of PBGS was constructed using Chlorobium vibrioform (Protein Data Bank 1W1Z) as a template by homology modeling method. RESULTS: The built structure was close to the crystal structure with Z score - 8.97. Molecular docking of DVDs into the S. aureus PBGS active site revealed that they are showing strong interaction forming H-bonds with the active sites of K248 and R217. The ligand-receptor complex of DVD13 showed a best docking score of - 14.4555 kcal/mol among DV and all its analogs while the substrate showed docking score of - 13.0392 kcal/mol showing interactions with S199, K217 indicating that DVD13 can influence structural variations on the enzyme and thereby inhibiting the enzyme. CONCLUSION: The substrate analog DVD13 is showing significant interactions with active site of PBGS and it may be used as a potent inhibitor to control S. aureus infections.

Identification of Microbial Pathogens in Periodontal disease and Diabetic patients of South Indian Population.

Periodontitis have been referred to as the sixth complication of diabetes found in high prevalence among diabetic patients than among healthy controls. The aim of the present study was to examine the periodontal disease status among collected dental plaque samples. Chromosomal DNA was isolated and amplified by universal primers. The DNA was sequenced for bacterial confirmation and phylogenetic analysis performed for the evolutionary relationship with other known pathogens. No amplification products were observed in groups labeled non periodontal and non Diabetes (NP&ND) and non Periodontal and Diabetes (NP&D). But in the case of Periodontal and non Diabetes (P&ND) groups 22 amplified products were observed. In case of Periodontal and Diabetes (P&D), 32 amplified products were positive for microbes. Among the four microbial groups, Treponemadenticola, and Tannerella forsythia were found to be prevalent in P&D. The phylogenetic analysis of 16s rRNA of Treponemadenticola showed the relationship with other Treponema oral pathogen species and with the Spirochaetazuelaera. Tannerella forsythia shows its evolutionary relationship only with four oral pathogens (Macellibacteroidesfermentans, Porphyromadaceae bacterium, Parabacteroidesmeredae and Bacillus fosythus). Prevotellaintermedia also showed its evolutionary relationship only with Prevotella Spcs while Fusobacterium revealed close evolutionary relationship only with Porpiromonasgingivalis.

Structural and Functional analysis of Staphylococcus aureus NADP-dependent IDH and its comparison with Bacterial and Human NADPdependent IDH.

Staphylococcus aureus a natural inhabitant of nasopharyngeal tract mainly survives as biofilms and possess complete Krebs cycle which plays major role in its pathogenesis. This TCA cycle is regulated by Isocitrate dehydrogenase (IDH) we have earlier cloned, sequenced (HM067707), expressed and characterized this enzyme from S. aureus ATCC12600. We have observed only one type of IDH in all the strains of S. aureus which dictates the flow of carbon thereby controlling the virulence and biofilm formation, this phenomenon is variable among bacteria. Therefore in the present study comparative structural and functional analysis of IDH was undertaken. As the crystal structure of S. aureus IDH was not available therefore using the deduced amino sequence of complete gene the 3D structure of IDH was built in Modeller 9v8. The PROCHECK and ProSAweb analysis showed the built structure was close to the crystal structure of Bacillus subtilis. This structure when superimposed with other bacterial IDH structures exhibited extensive structural variations as evidenced from the RMSD values correlating with extensive sequential variations. Only 24% sequence identity was observed with both human NADP dependent IDHs (PDB: 1T09 and 1T0L) and the structural comparative studies indicated extensive structural variations with an RMSD values of 14.284Å and 10.073Å respectively. Docking of isocitrate to both human IDHs and S. aureus IDH structures showed docking scores of -11.6169 and -10.973 respectively clearly indicating higher binding affinity of isocitrate to human IDH.

Isolation and characterization of ethanol tolerant yeast strains.

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.

Molecular characterization of α-amylase from Staphylococcus aureus.

Staphylococcus aureus is one of the prominent Gram positive human pathogen secretes many surface and secretary proteins including various enzymes and pathogenic factors that favour the successful colonization and infection of host tissue. α-amylase is one of the enzymes secreted by S. aureus which catalyses the breakdown of complex sugars to monosaccharides, which are required for colonization and survival of this pathogen in any anatomical locales. In the present study we have cloned, sequenced, expressed and characterized α-amylase gene from S. aureus ATCC12600. The recombinant enzyme has a molecular weight of 58kDa and the kinetics showed Vmax 0.0208±0.033 (mg/ml)/mg/min and Km 10.633±0.737mg/ml. The multiple sequence analysis showed α- amylase of S. aureus exhibited large differences with Bacillus subtilis and Streptococcus bovis. As the crystal structure of S. aureus α- amylase was unavailable, we used homology modelling method to build the structure. The built structure was validated by Ramachandran plot which showed 90% of the residues in the allowed region while no residue was found in the disallowed region and the built structure was close to the crystal structure with Z-Score: -6.85. The structural superimposition studies with α- amylases of Bacillus subtilis and Streptococcus bovis showed distinct differences with RMSD values of 18.158Åand 7.091Å respectively which correlated with enzyme kinetics, indicating α-amylase is different among these bacteria.

Cloning, expression and characterization of glucokinase gene involved in the glucose-6- phosphate formation in Staphylococcus aureus.

Glucose-6-phosphate (G-6-P) formation in Staphylococcus aureus is catalysed by glucokinase (glkA) gene under high glucose concentration leading to upregulation of various pathogenic factors; therefore the present study is aimed in the cloning and characterization of glk A gene from S. aureus ATCC12600. The glk A gene was cloned in the Sma I site of pQE 30, sequenced (Accession number: JN645812) and expressed in E. coli DH5α. The recombinant glk A expressed from the resultant glk A 1 clone was purified using nickel metal chelate chromatography, the pure enzyme gave single band in SDS-PAGE with molecular weight of 33kDa. The rglk A showed very high affinity to glucose Km 5.1±0.06mM with Hill coefficient of 1.66±0.032mM. Analysis of glucokinase sequence of S. aureus showed presence of typical ATP binding site and ROK motif CNCGRSGCIE. Sequentially and phylogenetically S. aureus glk A exhibited low identity with other bacterial glk A and 21% homology with human glucokinase (GCK). Functionally, S. aureus glk A showed higher rate of G-6-P formation compared to human GCK which may have profound role in the pathogenesis.

Comparison and correlation of binding mode of ATP in the kinase domains of Hexokinase family.

Hexokinases (HKs) are the enzymes that catalyses the ATP dependent phosphorylation of Hexose sugars to Hexose-6-Phosphate (Hex-6-P). There exist four different forms of HKs namely HK-I, HK-II, HK-III and HK-IV and all of them share a common ATP binding site core surrounded by more variable sequence that determine substrate affinities. Although they share a common binding site but they differ in their kinetic functions, hence the present study is aimed to analyze the binding mode of ATP. The analysis revealed that the four ATP binding domains are showing 13 identical, 7 similar and 6 dissimilar residues with similar structural conformation. Molecular docking of ATP into the kinase domains using Molecular Operating Environment (MOE) soft ware tool clearly showed the variation in the binding mode of ATP with variable docking scores. This probably explains the variable phosphorylation rates among hexokinases family.