Mechanistic insights into granule-bound starch synthase I (GBSSI.L539P) allele in high amylose starch biosynthesis in wheat (Triticum aestivum L.).

Amylose fraction of grain starch is correlated with a type of resistant starch with better nutritional quality. Granule-bound starch synthase I (GBSSI) is the known starch synthase, responsible for elongation of linear amylose chains. GBSSI expression, activity, and binding to starch and other proteins are the key factors that can affect amylose content. Previously, a QTL, qhams7A.1 carrying GBSSI mutant allele, was identified through QTL mapping using F2 population of the high amylose mutant line, 'TAC 75'. This high amylose mutant line has >2-fold higher amylose content than wild variety 'C 306'. In this study, we characterized this novel mutant allele, GBSSI.L539P. In vitro starch synthase activity of GBSSI.L539P showed improved activity than the wild type (GBSSI-wt). When expressed in yeast glycogen synthase mutants (Δgsy1gsy2), GBSSI-wt and GBSSI.L539P partially complemented the glycogen synthase (gsy1gsy2) activity in yeast. Structural analysis by circular dichroism (CD) and homology modelling showed no significant structural distortion in the mutant enzyme. Molecular docking studies suggested that the residue Leu539 is distant from the catalytic active site (ADP binding pocket) and had no detectable conformational changes in active site. Both wild and mutant enzymes were assayed for starch binding in vitro, and demonstrating higher affinity of the GBSSI.L539P mutant for starch than the wild type. The present study indicated that distant residue (L539P) influenced GBSSI activity by affecting its starch-binding ability. Therefore, it may be a potential molecular target for enhanced amylose content in grain.

Structural exploration of glutamine synthetase from Leishmania donovani: Insights from in silico and in vitro analysis.

Glutamine synthetase from L. donovani (LdGS) has been identified as a potential antileishmanial target in our previous report based on biochemical and inhibition studies. With the aim to structurally explore LdGS, systematic in silico and in vitro studies have been employed in the present study to identify amino acids crucial for LdGS mediated catalysis. A comparative analysis with human GS (HsGS) was performed which revealed significant differences in the active site pocket of human and parasite GS enzyme. The important amino acids identified from the in silico analysis of the optimized complexes, were subjected to in silico and in vitro alanine scanning by site directed mutagenesis. The results indicated crucial conserved and non conserved residues required for GS activity. The role of these residues in maintenance of secondary and tertiary structure of GS enzyme was also explored. In silico virtual screening was performed which resulted in the identification of five hits i.e. ZINC83236243, ZINC77319454, ZINC83236244, ZINC83236734 and ZINC83236736, as potential LdGS selective inhibitors. The illustrated structural and functional details of enzyme provides a better understanding of the structural integrity of LdGS and can be further utilized for the development of parasite specific GS inhibitors for treatment of visceral leishmaniasis infections.

Exploration of the expeditious potential of Pseudomonas fluorescens lipase in the kinetic resolution of racemic intermediates and its validation through molecular docking.

A profoundly time-efficient chemoenzymatic method for the synthesis of (S)-3-(4-chlorophenoxy)propan-1,2-diol and (S)-1-chloro-3-(2,5-dichlorophenoxy)propan-2-ol, two important pharmaceutical intermediates, was successfully developed using Pseudomonas fluorescens lipase (PFL). Kinetic resolution was successfully achieved using vinyl acetate as acylating agent, toluene/hexane as solvent, and reaction temperature of 30°C giving high enantioselectivity and conversion. Under optimized condition, PFL demonstrated 50.2% conversion, enantiomeric excess of 95.0%, enantioselectivity (E = 153) in an optimum time of 1 hour and 50.3% conversion, enantiomeric excess of 95.2%, enantioselectivity (E = 161) in an optimum time of 3 hours, for the two racemic alcohols, respectively. Docking of the R- and S-enantiomers of the intermediates demonstrated stronger H-bond interaction between the hydroxyl group of the R-enantiomer and the key binding residues of the catalytic site of the lipase, while the S-enantiomer demonstrated lesser interaction. Thus, docking study complemented the experimental outcome that PFL preferentially acylated the R form of the intermediates. The present study demonstrates a cost-effective and expeditious biocatalytic process that can be applied in the enantiopure synthesis of pharmaceutical intermediates and drugs.

Pharmacoinformatic Study on the Selective Inhibition of the Protozoan Dihydrofolate Reductase Enzymes.

Dihydrofolate reductase (DHFR) is an essential enzyme of the folate metabolic pathway in protozoa and it is a validated, potential drug target in many infectious diseases. Information about unique conserved residues of the DHFR enzyme is required to understand residual selectivity of the protozoan DHFR enzyme. The three dimensional crystal structures are not available for all the protozoan DHFR enzymes. Enzyme-substrate/inhibitor interaction information is required for the binding mode characterization in protozoan DHFR for selective inhibitor design. In this work, multiple sequence analysis was carried out in all the studied species. Homology models were built for protozoan DHFR enzymes, for which 3D structures are not available in PDB. The molecular docking and Prime-MMGBSA calculations of the natural substrate (dihydrofolate, DHF) and classical DHFR inhibitor (methotrexate, MTX) were performed in protozoan DHFR enzymes. Comparative sequence analysis showed that an overall sequence identity between the studied species ranging from 22.94 % (CfDHFR-BgDHFR) to 94.61 % (LdDHFR-LmDHFR). Interestingly, it was observed that most of the active site residues were conserved in all the cases and all the enzymes exhibit similar key binding interactions with DHF and MTX in molecular docking analysis, but there are a few key binding residues which differ in protozoan species that makes it suitable for target selectivity. This information can be used to design selective and potent protozoan DHFR enzyme inhibitors.

Prepulse minimization in KALI-5000.

A pulse power system (1 MV, 50 kA, and 100 ns) based on Marx generator and Blumlein pulse forming line has been built for generating high power microwaves. The Blumlein configuration poses a prepulse problem and hence the diode gap had to be increased to match the diode impedance to the Blumlein impedance during the main pulse. A simple method to eliminate prepulse voltage using a vacuum sparkgap and a resistor is given. Another fundamental approach of increasing the inductance of Marx generator to minimize the prepulse voltage is also presented. Experimental results for both of these configurations are given.