Computational Analysis of Thermal Adaptation in Extremophilic Chitinases: The Achilles' Heel in Protein Structure and Industrial Utilization.

Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a "weak link" from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop "hot spots" for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure-stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions.

Free radical scavenging activity in in vitro-derived tissues of Eruca sativa.

Feasible regeneration protocol for economically important plant Eruca sativa was established and 1, 1-diphenyl-2-picrylhydrazyl scavenging activity of regenerated tissues was evaluated and compared with plant material collected from the wild. Leaf portions inoculated onto Murashige and Skoog (MS) medium responded to all plant growth regulators exploited. Optimum callus production was achieved on a combination of 2.0 mg l(-1) 6-benzyladenine (BA) + 1.0 mg l(-1) α-naphthalene acetic acid (NAA) and the lowest response was recorded for 0.5 mg l(-1) gibberellic acid (GA3) + 1.0 mg l(-1) NAA. The callus was subcultured on similar composition/concentrations of plant growth regulators after 4 weeks of culture time. A 5.0 mg l(-1) 6-BA + 1.0 mg l(-1) NAA produced optimum percentage shoot organogenesis after 4 weeks of subculturing. However, optimum number of shoots per explant was recorded for moderate concentrations (1.0 and 2.0 mg l(-1)) of kinetin. Incorporation of NAA into MS medium-containing GA3 also produced a feasible number of shoots/explant. Similar mean shoot length was recorded for 2.0 mg l(-1) kinetin + 1.0 mg l(-1) NAA and optimum concentrations (2.0, 5.0, and 10.0 mg l(-1)) of GA3 + 1.0 mg l(-1) NAA. In vitro generated shoots were shifted to MS medium augmented with indole acetic acid (IAA) for rooting after 4 weeks of subculturing. Moderate concentrations (5.0 mg l(-1)) of IAA produced feasible rooting. Investigation of radical scavenging activity showed that callus possesses higher levels of radical scavengers than other plant tissues tested. Phenolics and glucosides are reported to be active components of Eruca sativa phytochemistry.

Coupling of surface carboxyls of carboxymethylcellulase with aniline via chemical modification: extreme thermostabilization in aqueous and water-miscible organic mixtures.

We wish to report the attainment of the highest ever T(opt) by introducing approximately two aromatic rings through chemical modification of surface carboxyl groups in carboxymethylcellulase from Scopulariopsis sp. with concomitant decrease in V(max), K(m), and optimum pH! This extraordinary enhancement in thermophilicity of aniline-coupled CMCase (T(opt) = 122 degrees C) by a margin of 73 degrees C as compared with the native enzyme (T(opt) = 49 degrees C) is the highest reported for any mesophilic enzyme that has been modified either through chemical modification or site-directed mutagenesis. It is also reported for the first time that aniline coupled CMCase (ACC) is simultaneously thermostable in aqueous as well as water-miscible organic solvents. The T(opt) of native CMCase and ACC were 25 and 90 degrees C, respectively, in 40% (v/v) aqueous dioxan. The modified enzyme was also stabilized against irreversible thermal denaturation. Therefore, at 55 degrees C, ACC had a half-life of 136 min as compared with native CMCase whose half-life was only 5 min. We believe that the reasons for this elevated thermostability and thermophilicity are surface aromatic-aromatic interactions and aromatic interactions with the sugar backbone of the substrate, respectively.

Comparative proteomics analysis of the root apoplasts of rice seedlings in response to hydrogen peroxide.

BACKGROUND: Plant apoplast is the prime site for signal perception and defense response, and of great importance in responding to environmental stresses. Hydrogen peroxide (H(2)O(2)) plays a pivotal role in determining the responsiveness of cells to stress. However, how the apoplast proteome changes under oxidative condition is largely unknown. In this study, we initiated a comparative proteomic analysis to explore H(2)O(2)-responsive proteins in the apoplast of rice seedling roots. METHODOLOGY/PRINCIPAL FINDINGS: 14-day-old rice seedlings were treated with low concentrations (300 and 600 µM) of H(2)O(2) for 6 h and the levels of relative electrolyte leakage, malondialdehyde and H(2)O(2) were assayed in roots. The modified vacuum infiltration method was used to extract apoplast proteins of rice seedling roots, and then two-dimensional electrophoresis gel analysis revealed 58 differentially expressed protein spots under low H(2)O(2) conditions. Of these, 54 were successfully identified by PMF or MS/MS as matches to 35 different proteins including known and novel H(2)O(2)-responsive proteins. Almost all of these identities (98%) were indeed apoplast proteins confirmed either by previous experiments or through publicly available prediction programs. These proteins identified are involved in a variety of processes, including redox homeostasis, cell wall modification, signal transduction, cell defense and carbohydrate metabolism, indicating a complex regulative network in the apoplast of seedling roots under H(2)O(2) stress. CONCLUSIONS/SIGNIFICANCE: The present study is the first apoplast proteome investigation of plant seedlings in response to H(2)O(2) and may be of paramount importance for the understanding of the plant network to environmental stresses. Based on the abundant changes in these proteins, together with their putative functions, we proposed a possible protein network that provides new insights into oxidative stress response in the rice root apoplast and clues for the further functional research of target proteins associated with H(2)O(2) response.

Proteomic response of rice seedling leaves to elevated CO2 levels.

Previous investigations of plant responses to higher CO 2 levels were mostly based on physiological measurements and biochemical assays. In this study, a proteomic approach was employed to investigate plant response to higher CO 2 levels using rice as a model. Ten-day-old seedlings were progressively exposed to 760 ppm, 1140 ppm, and 1520 ppm CO 2 concentrations for 24 h each. The net photosynthesis rate ( P n), stomatal conductance ( G s), transpiration rate ( E), and intercellular to ambient CO 2 concentration ratio ( C i/ C a) were measured. P n, G s, and E showed a maximum increase at 1140 ppm CO 2, but further exposure to 1520 ppm for 24 h resulted in down regulation of these. Proteins extracted from leaves were subjected to 2-DE analysis, and 57 spots showing differential expression patterns, as detected by profile analysis, were identified by MALDI-TOF/TOF-MS. Most of the proteins belonged to photosynthesis, carbon metabolism, and energy pathways. Several molecular chaperones and ascorbate peroxidase were also found to respond to higher CO 2 levels. Concomitant with the down regulation of P n and G s, the levels of enzymes of the regeneration phase of the Calvin cycle were decreased. Correlations between the protein profiles and the photosynthetic measurements at the three CO 2 levels were explored.