Molecular characterization of the thermostability and carbohydrate-binding module from a newly identified GH118 family agarase,AgaXa

AgaXa is a thermostable β-agarase from the agar-degrading bacterium Catenovulum sp. X3. To further understand the mechanism of protein stabilization of AgaXa, several mutants were generated by random and site-directed mutagenesis, and they were subsequently screened by analysing their enzymatic activity and thermostability. Four mutants (V197D, P259H, C442S and C528S) were found for which the enzyme activity and thermostability were significantly decreased. Moreover, secondary structures determined by circular dichroism (CD) showed that mutants V197D and P259H presented a higher percentage of α-helix but fewer β-strands than wild-type (WT) AgaXa. On the contrary, no significant changes were observed in the secondary structures of mutants C442S and C528S. Through the treatment by proteinase K, different digested profiles were generated from mutants V197D and P259H when compared to WT, and mutants C442S and C528S was observed with more digested protein fragments. These results indicate that the enzymatic activity and stability of AgaXa is mainly related to its hydrophobic structure and disulphide bonds. Furthermore, carbohydrate-binding ability was also analysed for the mutants of N- and C-terminal deletions, and the results showed that agarose binding capability was not changed, indicating that the carbohydrate-binding module is probably located in the middle of the β-agarase AgaXa.     

k-卡拉胶酶CgkX催化区的重组表达菌株筛选和发酵优化

目的：CgkX 是来自QY203 的一种高活性、高稳定性的k-卡拉胶酶，本文旨在构建CgkX 催化结构域 （CgkX-CM）重组表达菌株，并对发酵条件进行优化，提高CgkX-CM 的产量。方法：在分析k- 卡拉胶酶CgkX-CM基因序列的基 础上，构建多种CgkX-CM 表达载体，在大肠杆菌BL21（DE3）中进行重组表达，通过酶活测定筛选其中产量最高的表达菌株，并 优化发酵起始pH、诱导温度、IPTG浓度、装液量以及甘氨酸浓度等因素。结果：构建了5 种重组表达菌株，其中BL21 (DE3)/pET22b-CgkX-CM 酶产量是其他重组菌株的7.4-11.6 倍。确定了该菌株最佳发酵条件为：培养基含1 g·L-1甘氨酸（起始 pH 7.5），装液量为75 mL，0.1 mmol·L-1 IPTG 20 ℃诱导28 h，酶产量是优化前的7.3 倍。结论：经过重组表达载体筛选和发酵优 化，CgkX-CM产量大幅度提高，为今后比较CgkX 全长及其催化区域的酶学性质，确定C 端Big_2 结构域对CgkX的作用奠定了 基础。     

CBM在多糖底物降解中的应用进展

碳水化合物结合模块(carbohydrate-binding module, CBM)是碳水化合物活性酶的重要组成部分，其功能是识别并结合到特定的多糖底物上以提高催化结构域在底物附近的浓度及催化效率，帮助其更好地降解如纤维素、木聚糖、几丁质和黄原胶等大分子化合物。不同家族的CBM因其来源或结构不同往往会具有不同的底物结合特性。本文从CBM的家族、结构和功能等方面对CBM近年来的研究进行了综述，特别是对其作为融合单元运用到多糖底物的降解和糖苷水解酶改造方面的应用进行了总结。     

The nature of the carbohydrate binding module determines the catalytic efficiency of xylanase Z of Clostridium thermocellum

Xylanase Z of Clostridium thermocellum exists as a complex in the cellulosome with N-terminus feruloyl esterase, a carbohydrate binding module (CBM6) and a dockerin domain. To study the role of the binding modules on the activity of XynZ, different variants with the CBM6 attached to the catalytic domain at its C-terminal (XynZ-CB) and N-terminal (XynZ-BC), and the CBM22 attached at N-terminus (XynZ-B′C) were expressed in Escherichia coli at levels around 30% of the total cell proteins. The activities of XynZ-BC, XynZ-CB and XynZ-B′C were 4200, 4180 and 20,700 U μM⁻¹ against birchwood xylan, respectively. Substrate binding studies showed that in case of XynZ-BC and XynZ-CB the substrate birchwood xylan remaining unbound were 51 and 52%, respectively, whereas in the case of XynZ-B′C the substrate remaining unbound was 39% under the assay conditions used. The molecular docking studies showed that the binding site of CBM22 in XynZ-B′C is more exposed and thus available for substrate binding as compared to the tunnel shape binding pocket produced in XynZ-BC and thus hindering the substrate binding. The substrate binding data for the two constructs are in agreement with this explanation.     

Relationship between Superprecipitating Activity and Constituents of Myosin B Prepared from Normal and PSE Porcine Muscle

Myosin B from normal and PSE porcine muscles was studied by superprecipitation and SDS polyacrylamide gel electrophoresis. Similar studies were made on the fractions derived from the myosin B preparation after ultracentrifugation. Myosin B and its fractions from PSE muscle showed much less superprecipitation than normal. This difference between normal and PSE muscle seems to reflect differences in biological activity of their myofibrillar proteins, rather than differences in myofibrillar protein composition.     

Molecular Evolution of Multisubunit RNA Polymerases: Structural Analysis

Comprehensive multiple sequence alignments of the multisubunit DNA-dependent RNA polymerase (RNAP) large subunits, including the bacterial β and β′ subunits and their homologs from archaebacterial RNAPs, eukaryotic RNAPs I-III, nuclear-cytoplasmic large double-stranded DNA virus RNAPs, and plant plastid RNAPs, were created [Lane, W. J. and Darst, S. A. (2009). Molecular evolution of multisubunit RNA polymerases: sequence analysis. In press]. The alignments were used to delineate sequence regions shared among all classes of multisubunit RNAPs, defining common, fundamental RNAP features as well as identifying highly conserved positions. Here, we present a systematic, detailed structural analysis of these shared regions and highly conserved positions in terms of the RNAP structure, as well as the RNAP structure/function relationship, when known.     

The efficacy of the antibacterial peptide,pyrrhocoricin, is finely regulated by its amino acid residues and active domains

Summary Pyrrhocoricin, a highly active antibacterial peptide isolated from insects, inhibits chaperone-assisted protein folding via binding to the 70 kDa heat shock protein DnaK with its amino terminal half. The C-terminus functions as an intracellular delivery module. In the current study, chimeras consisting of the putative functional units of pyrrhocoricin and a related peptide, drosocin, were made, and it was found that some mixed and matched sequences retained their ability to killEscherichia coli, Salmonella typhimurium andAgrobacterium tumefaciens. While pyrrhocoricin appeared to have a more universal pharmacophore, drosocin featured a more robust intracellular delivery unit. We also identified the minimal length of pyrrhocoricin that is needed to efficiently kill bacteria. While for activity againstS. typhimurium the peptide could not be shortened, againstE. coli it was sufficient to have a Vall-Ile16 amino-terminal fragment. Although Vall was not part of the Asp2-Pro 10 pharmacophore (it could be replaced with other residues), it could not be eliminated and apparently played an important role in defining the activity of the peptide. Indeed, when Val1 was replaced with lysine, not only the efficacy of pyrrhocoricin to kill the sensitive strains increased significantly, resulting in the most active antimicrobial peptide against some clinical strains ever made, but the modified peptide was also able to killPseudomonas aeruginosa, an originally unresponsive bacterium in the low μg ml⁻¹ concentration range. However, this substitution likely influenced the interaction with bacterial membranes rather than that with the target protein, and therefore the dominant mode of action of the Lysl-pyrrhocoricin peptide may feature membrane disintegration instead of DnaK inhibition.     

Systematic prediction of autophagy-related proteins using Arabidopsis thaliana interactome data

Autophagy is a self-degradative process that is crucial for maintaining cellular homeostasis by removing damaged cytoplasmic components and recycling nutrients. Such an evolutionary conserved proteolysis process is regulated by the autophagy-related (Atg) proteins. The incomplete understanding of plant autophagy proteome and the importance of a proteome-wide understanding of the autophagy pathway prompted us to predict Atg proteins and regulators in Arabidopsis. Here, we developed a systems-level algorithm to identify autophagy-related modules (ARMs) based on protein subcellular localization, protein–protein interactions, and known Atg proteins. This generates a detailed landscape of the autophagic modules in Arabidopsis. We found that the newly identified genes in each ARM tend to be upregulated and coexpressed during the senescence stage of Arabidopsis. We also demonstrated that the Golgi apparatus ARM, ARM13, functions in the autophagy process by module clustering and functional analysis. To verify the in silico analysis, the Atg candidates in ARM13 that are functionally similar to the core Atg proteins were selected for experimental validation. Interestingly, two of the previously uncharacterized proteins identified from the ARM analysis, AGD1 and Sec14, exhibited bona fide association with the autophagy protein complex in plant cells, which provides evidence for a cross-talk between intracellular pathways and autophagy. Thus, the computational framework has facilitated the identification and characterization of plant-specific autophagy-related proteins and novel autophagy proteins/regulators in higher eukaryotes.