Recombinant production and biochemical and in silico characterization of lactate dehydrogenase from Geobacillus thermodenitrificans DSM-465

Background: Lactate dehydrogenase (LDH) is an enzyme of glycolytic pathway, ubiquitously found in living organisms. Increased glycolysis and LDH activity are associated with many pathologic conditions including inflammation and cancer, thereby making the enzyme a suitable drug target. Studies on conserved structural and functional domains of LDH from various species reveal novel inhibitory molecules. Our study describes Escherichia coli production and characterization of a moderately thermostable LDH (LDH-GT) from Geobacillus thermodenitrificans DSM-465. An in silico 3D model of recombinant enzyme and molecular docking with a set of potential inhibitors are also described. Results: The recombinant enzyme was overexpressed in E. coli and purified to electrophoretic homogeneity. The molecular weight of the enzyme determined by MALDI-TOF was 34,798.96 Da. It exhibited maximum activity at 65°C and pH 7.5 with a KM value for pyruvate as 45 µM. LDH-GT and human LDH-A have only 35.6% identity in the amino acid sequence. On the contrary, comparison by in silico structural alignment reveals that LDH-GT monomer has approximately 80% identity to that of truncated LDH-A. The amino acids "GEHGD" as well as His179 and His193 in the active site are conserved. Docking studies have shown the binding free energy changes of potential inhibitors with LDH-A and LDH-GT ranging from −407.11 to −127.31 kJ mol−1 . Conclusions: By highlighting the conserved structural and functional domains of LDH from two entirely different species, this study has graded potential inhibitory molecules on the basis of their binding affinities so that they can be applied for in vivo anticancer studies

Investigation of Genetic Diversity among Medicago species Using RAPD Markers.

Aims: Medicago is known as the Queen of forage with potential economic importance to our society. The present study aimed at the use of RAPD-PCR DNA marker to identify the genetic fingerprints affinities of six species of Alfalfa. Place and Duration of Study: The study was conducted at the Department of Genetics, Garden Campus, Hazara University, Mansehra Pakistan during February, 2011 to August, 2013. Methodology: In this study, six species of Medicago namely TWAL (Tetraploid Wisconsin Alfalfa Line), Medicago arborea, Medicago falcata, Medicago sativa, Medicago lupulina and Medicago polymorpha were used to explore the diversity of alfalfa. Seven out of 120 decamers produced 34 polymorphic loci with 100% polymorphism to identify the different species of Medicago crop. The range of polymorphic loci was observed from 300 to 700 bp. Eleven species specific loci were generated by seven decamers. Primer B-18 generated single specific locus 700 bp against genomic DNA of M. lupulina and it is important to identify particular species of Alfalfa. The bivariate data were recorded as the presence of locus 1 and absence 0 and then this data was transferred into A and C respectively to make it suitable for DNAMAN software (version 5.2.2.0; Applied Biostatistics Inc). Moreover, cluster analysis was performed using sequence alignment and divergence function of the DNAMAN against the bivariate data collected from the products of decamers. All members clustered in a unique pattern except M. falcata and M. lupulina those shared 86% homology. Three distinct groups were observed during UPGMA (Unweighted pair Group Method with Arithmetic Mean). During the phylogenetic study, TWAL was observed to have genetic diversity from other five species of Alfalfa. Conclusion: So, the present study is enabling us to discriminate different species of Alfalfa and it could be useful to identify and authenticate different species of the same genus of medicinal important plant from the Flora of Pakistan.