Dengue protease inhibition activity of selected Malaysian medicinal herbs.

Dengue fever is one of major health problem around the world including Malaysia. It is caused by the arthropode-borne flavivirus and transmitted by the bite of the Aedes aegypti or Aedes albopictus mosquito infected with one of the four dengue virus serotypes (DENV-1, DENV-2, DENV-3, or DENV-4). In this study, a screening exercise of various Malaysian medicinal plants showed that the extracts of Lawsonia inermis, Dryobalanops aromatica, Punica granatum, Zizyphus jujuba Lam. and Zingiber zerumbet exhibited potent inhibitory activity against NS2B-NS3 serine protease. The methanol extracts of Dryobalanops aromatica showed inhibition of 99.70 % at concentration of 200 µg/mL with IC50 value of 0.30 ± 0.16 µg/mL.

Dengue protease inhibition activity of selected Malaysian medicinal herbs

@#Dengue fever is one of major health problem around the world including Malaysia. It is caused by the arthropode-borne flavivirus and transmitted by the bite of the Aedes aegypti or Aedes albopictus mosquito infected with one of the four dengue virus serotypes (DENV-1, DENV-2, DENV-3, or DENV-4). In this study, a screening exercise of various Malaysian medicinal plants showed that the extracts of Lawsonia inermis, Dryobalanops aromatica, Punica granatum, Zizyphus jujuba Lam. and Zingiber zerumbet exhibited potent inhibitory activity against NS2B-NS3 serine protease. The methanol extracts of Dryobalanops aromatica showed inhibition of 99.70 % at concentration of 200 μg/mL with IC50 value of 0.30 ± 0.16 μg/mL.

Understanding thermostability factors of Aspergillus niger PhyA phytase: a molecular dynamics study.

Molecular dynamics simulation was used to study the dynamic differences between native Aspergillus niger PhyA phytase and a mutant with 20 % greater thermostability. Atomic root mean square deviation, radius of gyration, and number of hydrogen bonds and salt bridges are examined to determine thermostability factors. The results suggest that, among secondary structure elements, loops have the most impact on the thermal stability of A. niger phytase. In addition, the location rather than the number of hydrogen bonds is found to have an important contribution to thermostability. The results also show that salt bridges may have stabilizing or destabilizing effect on the enzyme and influence its thermostability accordingly.

Kinetic and mutagenic evidence for the role of histidine residues in the Lycopersicon esculentum 1-aminocyclopropane-1-carboxylic acid oxidase.

The ACCO gene from Lycopersicon esculentum (tomato) has been cloned into the expression vector PT7-7. The highly expressed protein was recovered in the form of inclusion bodies. ACCO is inactivated by diethyl pyrocarbonate (DEPC) with a second-order rate constant of 170 M(-1) min(-1). The pH-inactivation rate data imply the involvement of an amino acid residue with a pK value of 6.05. The difference UV spectrum of the the DEPC-inactivated versus native ACCO showed a single peak at 242 nm indicating the modification of histidine residues. The inactivation was reversed by the addition of hydroxylamine to the DEPC-inactivated ACCO. Substrate/cofactor protection studies indicate that both iron and ACC bind near the active site, which contains histidine residues. Four histidines of ACCO were individually mutated to alanine and glycine. H39A is catalytically active, while H177A, H177G, H211A, H211G, H234A, and H234G are basically inactive. The results indicate that histidine residues 177, 211, and 234 may serve as ligands for the active-site iron of ACCO and/or may play some important structural or catalytic role.

Essential cysteines in 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase from Escherichia coli: analysis by chemical modification and site-directed mutagenesis.

The enzyme 3-deoxy-D-manno-octulosonic acid 8-phosphate synthase (EC 4.1. 2.16) (KDO 8-P synthase) that catalyzes the condensation of D-arabinose 5-phosphate (A 5-P) with phosphoenolpyruvate (PEP) to give 3-deoxy-D-manno-octulosonic acid 8-phosphate (KDO 8-P) and inorganic phosphate (Pi) was inactivated by the thiol-modifying reagents 5,5-dithiobis (2-nitrobenzoate) (DTNB) and methyl methanethiosulfonate (MMTS). Reaction of cloned native KDO 8-P synthase with DTNB correlated with modification of two of the four cysteine sulfhydryls per monomer of enzyme and total loss of enzymatic activity which could be partially restored by treatment with dithiothreitol (DTT). Cyanolysis of the DTNB-inactivated enzyme with KCN led to the elimination of 2 equiv of 5-thio-2-nitrobenzoate and partial recovery of activity. The presence of either substrate(s) or product(s) provided no protection against inactivation nor affected the number of cysteines modified, indicating that the cysteines modified are most likely not at the active site of KDO 8-P synthase. Titration of denatured enzyme with DTNB resulted in the modification of all four cysteines. After treatment of native enzyme with MMTS, no cysteines could be titrated with DTNB and no enzymatic activity could be detected. Treatment of the MMTS-inactivated KDO 8-P synthase with DTT resulted in restoration of enzymatic activity and the presence of two DTNB-titratable cysteine residues. Based on these observations and a report that KDO 8-P synthase is inactivated in a time-dependent manner with 3-bromopyruvate and that the substrate PEP protects against this inactivation, all four cysteines (38, 166, 206, and 249) were individually mutated to alanines via a modified PCR methodology. The C206A and C249A mutants were both enzymatically active with K(m) and Vmax values approximately identical to those of wild-type KDO 8-P synthase, and both native mutants reacted with DTNB to modify only one of the three remaining cysteine sulfhydryls per monomer of enzyme. Titration of denatured C206A and C249A mutants resulted in the modification of three cysteines. The C38A and C166A mutants were both for the most part enzymatically inactive. Titration of native C38A and C166A with DTNB resulted in modification of two cysteines while titration of the denatured mutant protein resulted in modification of the three remaining cysteines. Circular dichroism measurements of wild-type KDO 8-P synthase and the four C --> A mutants indicate modest but significant changes in the structure of the mutants. These results indicate that C206 and C249 in native KDO 8-P synthase are readily accessible to the modification reagent DTNB and therefore inactivation may result from structural changes in the DTNB-modified KDO 8-P synthase or blockage of access of substrates to the active site. The C38 and C166 in native KDO 8-P synthase are inaccessible to the modification reagent DTNB, indicating that they are located in the interior of KDO 8-P synthase, and loss of activity in the C38A and C166A mutants suggests their essentiality in the KDO 8-P synthase reaction.

Time-dependent inhibition of porcine kidney trehalase by aminosugars.

The inhibitory effects of various nitrogen-containing sugars on porcine kidney trehalase were studied. Validamycin A, validoxylamine A and MDL 25,637 were found to be potent, time-dependent inhibitors of the enzyme in vitro. The validoxylamine A-inhibited enzyme showed slow but reversible reactivation over time (t1/2 = 1.2 h). To our knowledge, this is the first report of time-dependent inhibition exhibited by either these particular aminosugars or a trehalase.