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Publication Metadata only In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates(2002-03-01) Rabuesak Khumthong; Chanan Angsuthanasombat; Sakol Panyim; Gerd Katzenmeier; Mahidol UniversityThe NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease... complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UVPublication Open Access Enzymatic analysis of recombinant Japanese encephalitis virus NS2B(H)-NS3pro protease with fluorogenic model peptide substrates(2012-05) Muhammad Junaid; Chakard Chalayut; Anna Sehgelmeble Torrejon; Chanan Angsuthanasombat; Iryna Shutava; Maris Lapins; Jarl E. S. Wikberg; Gerd Katzenmeier; Mahidol University. Institute of Molecular Biosciences. Laboratory of Molecular and Cellular Microbiology(SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblottingPublication Open Access Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3(2010) Wanisa Salaemae; Muhammad Junaid; Chanan Angsuthanasombat; Gerd Katzenmeier; Mahidol University. Institute of Molecular Biosciences. Laboratory of Molecular VirologyBackground The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed. Methods Alanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters Km, kcat and kcat/Km were determined. Results Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole. Conclusions Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.