Activity enhancement of a Bacillus circulans xylanase by introducing ion-pair interactions into an α-helix

IntroductionIt is widely accepted that ion-pair increases rigidity and thermostability. There are numerous studies on ion-pairs and thermostability, but none are available about the effect of ion-pair on the activity of enzymes. This paper studies whether an ion-pair allows flexible movement in an enzyme molecule and affects its activity.Materials and methodsIon-pairs are designed at the α-helix region of a Bacillus circulans xylanase, and they are far from the active-sites (23.85–25.15 Å). Two ion-pairing mutations are situated at the C-terminus (D151/E151-K154 ion-pairs) of the helix. One mutation is double-site (F48R-N151D), which introduces both the tertiary (R48-D151) and intra-helical (D151-K154) ion-pairs.Results and discussionAll of the mutants enhanced the catalytic efficiency against xylan (1.66–3.58 times). The double-site mutation showed a synergistic effect on the activity. Overall, the ion-pairs decreased the flexibility (increased rigidity) of the α-helix region and increased the active-site flexibility. The ion-pairs were destabilizing and surface-located; this means that the weaker destabilizing ion-pair still allows flexible movement in the active-site. There is higher mobility of the strand B4 where the active site residue E172 is located. Moreover, the residues lining the active-site cleft (strand B8) showed increased flexibility upon substrate binding.ConclusionIncrease in the activity was due to the increase in active-site flexibility and increased mobility of the residues lining the active-site cleft (strand B8).     

A novel thermostable cellulase free xylanase stable in broad range of pH from Streptomyces sp. CS428

A cellulase free thermostable xylanase from Streptomyces sp. CS428 was isolated from a Korean soil sample, purified by single-step chromatography, and biochemically characterized. The extracellular xylanase was purified 26 fold with a 55% yield by CM Trisacryl cation exchange chromatography. The molecular mass of the enzyme (Xyn428) was approximately 37 kDa. Xyn428 was found to be stable over a broad pH range (4 to ～13.6) and to 50 °C and have an optimum temperature of 80 °C. Xyn428 had K_m and V_max values of 102.3 ± 1.2 mg/mL and 3225.4 ± 15 mmol/min mg, respectively, when beechwood xylan was used as substrate. N-terminal sequence of Xyn428 was INRTDHNENSYLEIHNNEAR. CS428 was grown on different agro waste xylan and produced 4197.1 U/mL of xylanase activity in 36 h of cultivation in wheat bran without supplements. Xyn428 activity was inhibited by Tris salt at concentrations above 20 mM, and produced xylose and xylobiose as major products. It was found to degrade agro waste materials by small unit of enzyme (20 U/g) as shown by electron microscopy. As being simple in purification, thermo tolerant, pH stability in broad range and ability to produce xylooligosaccharides show that Xyn428 has potential applications in industries as a biobleaching agent and for xylooligosaccharides production.     

Fatty acid synthase inhibitor cerulenin inhibits topoisomerase I catalytic activity and augments SN-38-induced apoptosis

Fatty acid synthase (FASN) is overexpressed in a wide variety of human cancers, making it an attractive target for anticancer therapy. One of the most widely used inhibitors of FASN, cerulenin, is a natural product of Cephalosporium caerulens. Cerulenin is selectively toxic to human cancer cells in vitro. However, the mechanism by which FASN inhibition causes apoptosis in tumor cells remains unclear. Because of the widespread clinical interest in combining cerulenin with other chemotherapeutic agents, we performed this study to gain insight into the downstream effects of FASN inhibition that lead to apoptosis. Here, we observed the increased antitumor effect of cerulenin when combined with the topoisomerase inhibitor SN-38. We identified topoisomerase I as a potential mediator of cerulenin-induced apoptosis, possibly by upregulating intracellular polyunsaturation. Finally, we show that suppressing topoisomerase I catalytic activity results in synergistic effects between cerulenin and LY294002. Our results suggest that topoisomerase I could participate in cerulenin-induced apoptosis by upregulating intracellular polyunsaturation. These results will help determine the molecular basis of the cerulenin and SN-38 drug combination. Further investigation of this pathway will provide new insight into cancer cell metabolism and may aid in the design of additional cancer chemotherapeutic agents.     

Selective activation of the latissimus dorsi and the inferior fibers of trapezius at various shoulder angles during isometric pull-down exertion

The aim of this study was to determine the effect of isometric pull down exercise on muscle activity with shoulder elevation angles of 60°, 90°, and 120° and sagittal, scapular, and frontal movement planes, by electromyography (EMG) of the latissimus dorsi, inferior fibers of trapezius, and latissimus dorsi/inferior fibers of trapezius activity ratio. Fourteen men performed nine conditions of isometric pull down exercise (three conditions of shoulder elevation × three conditions of movement planes). Surface EMG was used to collect data from the latissimus dorsi and inferior fibers of trapezius during exercise. Two-way repeated analysis of variance with two within-subject factors (shoulder elevation angles and planes of movement) was used to determine the significance of the latissimus dorsi and inferior fibers of trapezius activity and latissimus dorsi/inferior fibers of trapezius activity ratio. The latissimus dorsi activity and ratio between the latissimus dorsi and the inferior fibers of trapezius were significantly decreased as shoulder elevation angle increased from 60° to 120°. The inferior fibers of trapezius activity was significantly increased with shoulder elevation angle. The EMG activity and the ratios were not affected by changes in movement planes. This study suggests that selective activation of the latissimus dorsi is accomplished with a low shoulder elevation angle, while the inferior fibers of the trapezius are activated with high shoulder elevation angles.     

Thermostabilization of Bacillus subtilis lipase A by minimizing the structural deformation caused by packing enhancement

Enzyme thermostabilization is a critical research topic due to potential industrial benefits. Among the various reasons to increase enzyme thermostability, enhancement of residual packing at the core of the enzyme structure has been commonly accepted as a successful strategy. However, structural changes that occur with residual packing enhancement may decrease enzyme activity. In this study, a strategy to minimize structural deformation by calculating the overlapping packing volume of a single-point mutation followed by applying a double-point mutation was suggested. Four double mutants, A38V_K23A, A75V_T83A, G80A_N106A, and G172A_V100A, were selected for the in vitro experiment; three of the four showed enhancements in both thermostability and catalytic activity. In particular, G80A_N106A showed 2.78 times higher catalytic activity compared with wild type.     

Genome sequence analysis of H5N1 influenza a virus isolated from a vietnamese in 2007

Highly pathogenic H5N1 avian influenza A virus (AIV) crossed the species barrier and caused a number of deaths in humans in Vietnam and 14 other countries. Since the last report of human H5N1 infection in November 2005, the first documented H5N1 human infection was reported in June 2007 in Vietnam and was followed by 7 more cases, including 5 fatalities. In this study, we isolated and analyzed the full length of the H5N1 genome from a sample from the first patient in 2007. Phylogenetic analysis of eight genomic segments of the H5N1 virus strain (A/Vietnam/HN/2007, VNH07) revealed that this strain appears to be of genotype V and contains the HA gene, which is classified into clade 2.3.4. The deduced amino acid sequence of the HA protein has a typical affinity sequence for α2,3 linkage (SAα2,3-Gal) receptors and typical multibasic cleavage sequences. Compared with other H5N1 isolates, VNH07 showed that the possible reassortments for the NA and NP segments occurred between A/goose/Guangxi/3017/2005-like iso?lates (2.3.2) and A/human/Zhejiang/16/2006-like isolates (2.3.4).     

Kaposi’s sarcoma-associated herpesvirus infection of endothelial progenitor cells impairs angiogenic activity <Emphasis Type="Italic">in vitro</Emphasis>

A recent study reported that endothelial progenitor cells (EPCs0 are one of the reservoirs of Kaposi’s sarcoma associated herpesvirus (KSHV). Although EPCs are closely linked to angiogenesis and vasculogenesis, little is known about the angiogenic potential of KSHV in EPCs. In this study, we used EPCs isolated from human umbilical cord blood to show that early infection by KSHV in vitro impaired the neovascularization of EPCs in matrigel. Our results suggest that KSHV may disrupt the angiogenic potential of EPCs and that the disseminated infection of KSHV could be associated with EPC dysfunction.