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The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30 degrees C and retaining more than 25% of its activity at 4 degrees C. The optimum pH was 8-8.5. The enzyme was active against short synthetic p-nitrophenylesters (C2-C10) with maximum activity towards the acetate ester (C2). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1-1.9).

Novel lipases lip11 and lip12 from Yarrowia lipolytica MSR80 were cloned and expressed in E. coli HB101 pEZZ18 system along with lip2. These enzymes were constitutively expressed as extracellular proteins with IgG tag. The enzymes were purified by affinity chromatography and analyzed by SDS-PAGE with specific activity of 314, 352 and 198 U/mg for Lip2, Lip11 and Lip12, respectively on olive oil. Biochemical characterization showed that all were active over broad range of pH 4.0-9.0 and temperature 20-80 °C with optima at pH 7 and 40 °C. All the three lipases were thermostable up to 80 °C with varying t(½). Activity on various substrates revealed that they were most active on oils > triacylglycerides > p-np-esters. Relatively Lip2 and Lip11 showed specificity for mid to long chain fatty acids, while Lip12 was mid chain specific. GC analysis of triolein hydrolysis by these lipases revealed that Lip2 and Lip11 are regioselective, while Lip12 is not. Effect of metal ions showed that Lip2 and Lip12 were activated by Ca²⁺ whereas Lip11 by Mg²⁺. All were thiol activated and inhibited by PMSF and N-bromosuccinimide. All were activated by non polar solvents and inhibited by polar solvents. Detailed sequence analysis and structural predictions revealed Lip11 and Lip12 shared 61 and 62 % homology with Lip2 (3O0D) and three dimensional superimposition revealed Lip2 was closer to Lip11 than to Lip12 as was observed during biochemical characterization. Finally, thermostability and substrate specificity has been explained on the basis of detailed amino acid analysis.

Backgroundγ-glutamyltranspeptidase (GGT) is a bi-substrate enzyme conserved in all three domains of life. It catalyzes the cleavage and transfer of γ-glutamyl moiety of glutathione to either water (hydrolysis) or substrates like peptides (transpeptidation). GGTs exhibit great variability in their enzyme kinetics although the mechanism of catalysis is conserved. Recently, GGT has been shown to be a virulence factor in microbes like Helicobacter pylori and Bacillus anthracis. In mammalian cells also, GGT inhibition prior to chemotherapy has been shown to sensitize tumors to the therapy. Therefore, lately both bacterial and eukaryotic GGTs have emerged as potential drug targets, but the efforts directed towards finding suitable inhibitors have not yielded any significant results yet. We propose that delineating the residues responsible for the functional diversity associated with these proteins could help in design of species/clade specific inhibitors.ResultsIn the present study, we have carried out phylogenetic analysis on a set of 47 GGT-like proteins to address the functional diversity. These proteins segregate into various subfamilies, forming separate clades on the tree. Sequence conservation and motif prediction studies show that even though most of the highly conserved residues have been characterized biochemically in previous studies, a significant number of novel putative sites and motifs are discovered that vary in a clade specific manner. Many of the putative sites predicted during the functional divergence type I and type II analysis, lie close to the known catalytic residues and line the walls of the substrate binding cavity, reinforcing their role in modulating the substrate specificity, catalytic rates and stability of this protein.ConclusionThe study offers interesting insights into the evolution of GGT-like proteins in pathogenic vs. non-pathogenic bacteria, archaea and eukaryotes. Our analysis delineates residues that are highly specific to each GGT subfamily. We propose that these sites not only explain the differences in stability and catalytic variability of various GGTs but can also aid in design of specific inhibitors against particular GGTs. Thus, apart from the commonly used in-silico inhibitor screening approaches, evolutionary analysis identifying the functional divergence hotspots in GGT proteins could augment the structure based drug design approaches.ReviewersThis article was reviewed by Andrei Osterman, Christine Orengo, and Srikrishna Subramanian. For complete reports, see the Reviewers’ reports sectionElectronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0080-7) contains supplementary material, which is available to authorized users.

Mouse double minute 2 (MDM2) protein functionally inactivates the tumor suppressor p53 in human cancer. Conventional MDM2 inhibitors provide limited clinical application as they interfere only with the MDM2-p53 interaction to release p53 from MDM2 sequestration but do not prevent activated p53 from transcriptionally inducing MDM2 expression. Here, we report a rationally synthesized chalcone-based pyrido[ b ]indole, CPI-7c, as a unique small-molecule inhibitor of MDM2, which not only inhibited MDM2-p53 interaction but also promoted MDM2 degradation. CPI-7c bound to both RING and N-terminal domains of MDM2 to promote its ubiquitin-mediated degradation and p53 stabilization. CPI-7c-induced p53 directly recruited to the promoters of DR4 and DR5 genes and enhanced their expression, resulting in sensitization of TNF-related apoptosis-inducing ligand (TRAIL)-resistant cancer cells toward TRAIL-induced apoptosis. Collectively, we identified CPI-7c as a novel small-molecule inhibitor of MDM2 with a unique two-prong mechanism of action that sensitized TRAIL-resistant cancer cells to apoptosis by modulating the MDM2-p53-DR4/DR5 pathway.

γ-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with γ-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni(2+)-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t (1/2) of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Ptγ-GT shared 37 % sequence identity and 53 % homology with an extremophile γ-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.

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Resveratrol enhances the BRCA1 gene expression and the MBD family of proteins bind to the promoter region of the BRCA1 gene.