COG3926 and COG5526: a tale of two new lysozyme-like protein families

We have identified two new lysozyme-like protein families by using a combination of sequence similarity searches, domain architecture analysis, and structural predictions. First, the P5 protein from bacteriophage phi8, which belongs to COG3926 and Pfam family DUF847, is predicted to have a new lysozyme-like domain. This assignment is consistent with the lytic function of P5 proteins observed in several related double-stranded RNA bacteriophages. Domain architecture analysis reveals two lysozyme-associated transmembrane modules (LATM1 and LATM2) in a few COG3926/DUF847 members. LATM2 is also present in two proteins containing a peptidoglycan binding domain (PGB) and an N-terminal region that corresponds to COG5526 with uncharacterized function. Second, structure prediction and sequence analysis suggest that COG5526 represents another new lysozyme-like family. Our analysis offers fold and active-site assignments for COG3926/DUF847 and COG5526. The predicted enzymatic activity is consistent with an experimental study on the zliS gene product from Zymomonas mobilis, suggesting that bacterial COG3926/DUF847 members might be activators of macromolecular secretion.     

Structure,Dynamics, and Specificity of Endoglucanase D from Clostridium cellulovorans

The enzymatic degradation of cellulose is a critical step in the biological conversion of plant biomass into an abundant renewable energy source. An understanding of the structural and dynamic features that cellulases utilize to bind a single strand of crystalline cellulose and hydrolyze the β-1,4-glycosidic bonds of cellulose to produce fermentable sugars would greatly facilitate the engineering of improved cellulases for the large-scale conversion of plant biomass. Endoglucanase D (EngD) from Clostridium cellulovorans is a modular enzyme comprising an N-terminal catalytic domain and a C-terminal carbohydrate-binding module, which is attached via a flexible linker. Here, we present the 2.1-Å-resolution crystal structures of full-length EngD with and without cellotriose bound, solution small-angle X-ray scattering (SAXS) studies of the full-length enzyme, the characterization of the active cleft glucose binding subsites, and substrate specificity of EngD on soluble and insoluble polymeric carbohydrates. SAXS data support a model in which the linker is flexible, allowing EngD to adopt an extended conformation in solution. The cellotriose-bound EngD structure revealed an extended active-site cleft that contains seven glucose-binding subsites, but unlike the majority of structurally determined endocellulases, the active-site cleft of EngD is partially enclosed by Trp162 and Tyr232. EngD variants, which lack Trp162, showed a significant reduction in activity and an alteration in the distribution of cellohexaose degradation products, suggesting that Trp162 plays a direct role in substrate binding.     

Gene coexpression analysis offers important modules and pathway of human lung adenocarcinomas

Lung adenocarcinomas injured greatly on the people worldwide. Although clinic experiments and gene profiling analyses had been well performed, to our knowledge, systemic coexpression analysis of human genes for this cancer is still limited to date. Here, using the published data GSE75037, we built the coexpression modules of genes by Weighted Gene Co-Expression Network Analysis (WGCNA), and investigated function and protein–protein interaction network of coexpression genes by Database for Annotation, visualization, and Integrated Discovery (DAVID) and String database, respectively. First, 11 coexpression modules were conducted for 5,000 genes in the 83 samples recently. Number of genes for each module ranged from 90 to 1,260, with the mean of 454. Second, interaction relationships of hub-genes between pairwise modules showed great differences, suggesting relatively high scale independence of the modules. Third, functional enrichment of the coexpression modules showed great differences. We found that genes in modules 8 significantly enriched in the biological process and/or pathways of cell adhesion, extracellular matrix (ECM)–receptor interaction, focal adhesion, and PI3K-Akt signaling pathway, and so forth. It was inferred as the key module underlying lung adenocarcinomas. Furthermore, PPI analysis revealed that the genes COL1A1, COL1A2, COL3A1, CTGF, and BGN owned the largest number of adjacency genes, unveiling that they may functioned importantly during the occurrence of lung adenocarcinomas. To summary, genes involved in cell adhesion, ECM–receptor interaction, focal adhesion, and PI3K-Akt signaling pathway play crucial roles in human lung adenocarcinomas.     

Preparation of Triacylglycerol Molecular Species by Interesterification Using Endocellular Lipase in n-Hexane

The interesterification of triacylglycerol with fatty acid was done to prepare triacylglycerol molecular species. Optimum operating conditions for the interesterification using a 1,3-positional specific endocellular lipase from Rhizopus japonicus NR400 in a batch system were investigated. The reaction was done at 40°C for 5 hr in the following system: Trioleoylglycerol-palmitic acid = 1:3.5 (mol/mol), 10 ml n-hexane/g trioleoylglycerol, and 2500 units of enzyme/g trioleoylglycerol. Under these conditions, the content of palmitoyl groups in 1,3-positions of triacylglycerol was about 60 mol%. Additional interesterification (2-cycle reaction) using palmitic acid and the novel triacylglycerol prepared by one-step interesterification (1-cycle reaction) resulted in a preparation of highly pure 1,3-dipalmitoyl-2-oleoylglycerol.     

Evaluation of the xylan breakdown potential of eight mesophilic endoxylanases

In biomass degradation using simultaneous saccharification and fermentation (SSF), there is a need for efficient biomass degrading enzymes that can work at lower temperatures suitable for yeast fermentation. As xylan is an important lignocellulosic biomass constituent, this study aimed at investigating the possible differences in xylan breakdown potential of endoxylanases using eight different endoxylanases at conditions relevant for SSF. Both solubilising and degrading capacities of the endoxylanases were investigated using water-insoluble and water-soluble oat spelt xylan as model substrates for biomass xylan. Results showed that selecting for combinations of endoxylanases that are efficient at solubilising xylan on the one hand and degrading it to large extent on the other hand, coupled to high specific activities, seems the best option for complete xylan breakdown in lignocellulosic biomass conversion using SSF.     

The structural basis of alpha-glucan recognition by a family 41 carbohydrate-binding module from Thermotoga maritima

Starch recognition by carbohydrate-binding modules (CBMs) is important for the activity of starch-degrading enzymes. The N-terminal family 41 CBM, TmCBM41 (from pullulanase PulA secreted by Thermotoga maritima) was shown to have alpha-glucan binding activity with specificity for alpha-1,4-glucans but was able to tolerate the alpha-1,6-linkages found roughly every three or four glucose units in pullulan. Using X-ray crystallography, the structures were solved for TmCBM41 in an uncomplexed form and in complex with maltotetraose and 6(3)-alpha-D-glucosyl-maltotriose (GM3). Ligand binding was facilitated by stacking interactions between the alpha-faces of the glucose residues and two tryptophan side-chains in the two main subsites of the carbohydrate-binding site. Overall, this mode of starch binding is quite well conserved by other starch-binding modules. The structure in complex with GM3 revealed a third binding subsite with the flexibility to accommodate an alpha-1,4- or an alpha-1,6-linked glucose.     

Walking through protein sequence space

Following the original idea of Maynard Smith on evolution of the protein sequence space, a novel tool is developed that allows the "space walk", from one sequence to its likely evolutionary relative and further on. At a given threshold of identity between consecutive steps, the walks of many steps are possible. The sequences at the ends of the walks may substantially differ from one another. In a sequence space of randomized (shuffled) sequences the walks are very short. The approach opens new perspectives for protein evolutionary studies and sequence annotation.     

Identification of genes encoding microbial glucuronoyl esterases

One type of covalent linkages connecting lignin and hemicellulose in plant cell walls is the ester linkage between 4-O-methyl-D-glucuronic acid of glucuronoxylan and lignin alcohols. An enzyme that could hydrolyze such linkages, named glucuronoyl esterase, occurs in the cellulolytic system of the wood-rotting fungus Schizophyllum commune. Here we report partial amino acid sequences of the enzyme and the results of subsequent search for homologous genes in sequenced genomes. The homologous genes of unknown functions were found in genomes of several filamentous fungi and one bacterium. The gene corresponding to the cip2 gene of Hypocrea jecorina (Trichoderma reesei), known to be up-regulated under conditions of induction of cellulolytic and hemicellulolytic enzymes, was over-expressed in H. jecorina. The product of the cip2 gene was purified to homogeneity and shown to exhibit glucuronoyl esterase activity.