Structural and biochemical analysis of family 92 carbohydrate-binding modules uncovers multivalent binding to ß-glucans.

Carbohydrate-binding modules (CBMs) are non-catalytic proteins found appended to carbohydrate-active enzymes. Soil and marine bacteria secrete such enzymes to scavenge nutrition, and they often use CBMs to improve reaction rates and retention of released sugars. Here we present a structural and functional analysis of the recently established CBM family 92. All proteins analysed bind preferentially to ß-1,6-glucans. This contrasts with the diversity of predicted substrates among the enzymes attached to CBM92 domains. We present crystal structures for two proteins, and confirm by mutagenesis that tryptophan residues permit ligand binding at three distinct functional binding sites on each protein. Multivalent CBM families are uncommon, so the establishment and structural characterisation of CBM92 enriches the classification database and will facilitate functional prediction in future projects. We propose that CBM92 proteins may cross-link polysaccharides in nature, and might have use in novel strategies for enzyme immobilisation.

Study on the Influence of Label Image Accuracy on the Performance of Concrete Crack Segmentation Network Models.

A high-quality dataset is a basic requirement to ensure the training quality and prediction accuracy of a deep learning network model (DLNM). To explore the influence of label image accuracy on the performance of a concrete crack segmentation network model in a semantic segmentation dataset, this study uses three labelling strategies, namely pixel-level fine labelling, outer contour widening labelling and topological structure widening labelling, respectively, to generate crack label images and construct three sets of crack semantic segmentation datasets with different accuracy. Four semantic segmentation network models (SSNMs), U-Net, High-Resolution Net (HRNet)V2, Pyramid Scene Parsing Network (PSPNet) and DeepLabV3+, were used for learning and training. The results show that the datasets constructed from the crack label images with pix-el-level fine labelling are more conducive to improving the accuracy of the network model for crack image segmentation. The U-Net had the best performance among the four SSNMs. The Mean Intersection over Union (MIoU), Mean Pixel Accuracy (MPA) and Accuracy reached 85.47%, 90.86% and 98.66%, respectively. The average difference between the quantized width of the crack image segmentation obtained by U-Net and the real crack width was 0.734 pixels, the maximum difference was 1.997 pixels, and the minimum difference was 0.141 pixels. Therefore, to improve the segmentation accuracy of crack images, the pixel-level fine labelling strategy and U-Net are the best choices.

Research on the Efficiency of Bridge Crack Detection by Coupling Deep Learning Frameworks with Convolutional Neural Networks.

Bridge crack detection based on deep learning is a research area of great interest and difficulty in the field of bridge health detection. This study aimed to investigate the effectiveness of coupling a deep learning framework (DLF) with a convolutional neural network (CNN) for bridge crack detection. A dataset consisting of 2068 bridge crack images was randomly split into training, verification, and testing sets with a ratio of 8:1:1, respectively. Several CNN models, including Faster R-CNN, Single Shot MultiBox Detector (SSD), You Only Look Once (YOLO)-v5(x), U-Net, and Pyramid Scene Parsing Network (PSPNet), were used to conduct experiments using the PyTorch, TensorFlow2, and Keras frameworks. The experimental results show that the Harmonic Mean (F1) values of the detection results of the Faster R-CNN and SSD models under the Keras framework are relatively large (0.76 and 0.67, respectively, in the object detection model). The YOLO-v5(x) model of the TensorFlow2 framework achieved the highest F1 value of 0.67. In semantic segmentation models, the U-Net model achieved the highest detection result accuracy (AC) value of 98.37% under the PyTorch framework. The PSPNet model achieved the highest AC value of 97.86% under the TensorFlow2 framework. These experimental results provide optimal coupling efficiency parameters of a DLF and CNN for bridge crack detection. A more accurate and efficient DLF and CNN model for bridge crack detection has been obtained, which has significant practical application value.

A polysaccharide utilization locus from Chitinophaga pinensis simultaneously targets chitin and ß-glucans found in fungal cell walls.

In nature, complex carbohydrates are rarely found as pure isolated polysaccharides. Instead, bacteria in competitive environments are presented with glycans embedded in heterogeneous matrices such as plant or microbial cell walls. Members of the Bacteroidota phylum thrive in such ecosystems because they are efficient at extracting nutrients from complex substrates, secreting consortia of synergistic enzymes to release metabolizable sugars. Carbohydrate-binding modules (CBMs) are used to target enzymes to substrates, enhancing reaction rate and product release. Additionally, genome organizational tools like polysaccharide utilization loci (PULs) ensure that the appropriate set of enzymes is produced when needed. In this study, we show that the soil bacterium Chitinophaga pinensis uses a PUL and several CBMs to coordinate the activities of enzymes targeting two distinct polysaccharides found in fungal cell walls. We describe the enzymatic activities and carbohydrate-binding behaviors of components of the fungal cell wall utilization locus (FCWUL), which uses multiple chitinases and one ß-1,3-glucanase to hydrolyze two different substrates. Unusually, one of the chitinases is appended to a ß-glucan-binding CBM, implying targeting to a bulk cell wall substrate rather than to the specific polysaccharide being hydrolyzed. Based on our characterization of the PUL's outer membrane sensor protein, we suggest that the FCWUL is activated by ß-1,3-glucans, even though most of its enzymes are chitin-degrading. Our data showcase the complexity of polysaccharide deconstruction in nature and highlight an elegant solution for how multiple different glycans can be accessed using one enzymatic cascade. IMPORTANCE We report that the genome of the soil bacterium Chitinophaga pinensis encodes three multi-modular carbohydrate-active enzymes that work together to hydrolyze the major polysaccharide components found in fungal cell walls (FCWs). The enzymes are all encoded by one polysaccharide utilization locus and are co-expressed, potentially induced in the presence of ß-1,3-glucans. We present biochemical characterization of each enzyme, including the appended carbohydrate-binding modules that likely tether the enzymes to a FCW substrate. Finally, we propose a model for how this so-called fungal cell wall utilization locus might enable C. pinensis to metabolize both chitin and ß-1,3-glucans found in complex biomass in the soil.

Family 92 carbohydrate-binding modules specific for ß-1,6-glucans increase the thermostability of a bacterial chitinase.

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide ß-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10-15 °C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

Multiple enzymatic approaches to hydrolysis of fungal ß-glucans by the soil bacterium Chitinophaga pinensis.

The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, ß-1,3-glucan and ß-1,6-glucan. While several fungal ß-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using ß-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the ß-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of ß-1,6-glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal ß-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates.

Integrity Assessment of Isolated Plant Mitochondria.

The integrity of isolated mitochondria can be estimated functionally using enzymatic activities or the permeability of mitochondrial membranes to molecules of different sizes. Thus, the permeability of the outer membrane to the protein cytochrome c, the permeability of the inner membrane to protons, and the permeability of the inner membrane to NAD+, NADH and organic acids using soluble matrix dehydrogenases as markers have all been used. These assays all have limitations to how the data can be converted into a measure of integrity, are differently sensitive to artifacts and require widely variable amounts of material. Therefore, each method has a restricted utility for estimating integrity, depending on the type of mitochondria analysed. Here, we review the advantages and disadvantages of different integrity assays and present protocols for integrity assays that require relatively small amounts of mitochondria. They are based on the permeability of the outer membrane to cytochrome c, and the inner membrane to protons or NAD(H). The latter has the advantage of utilizing a membrane-bound activity (complex I) and the pore-forming peptide alamethicin to gain access to the matrix space. These methods together provide a toolbox for the determination of functionality and quality of isolated mitochondria.

Mitochondrial NAD(P)H oxidation pathways and nitrate/ammonium redox balancing in plants.

Plant mitochondrial oxidative phosphorylation is characterised by alternative electron transport pathways with different energetic efficiencies, allowing turnover of cellular redox compounds like NAD(P)H. These electron transport chain pathways are profoundly affected by soil nitrogen availability, most commonly as oxidized nitrate (NO3-) and/or reduced ammonium (NH4+). The bioenergetic strategies involved in assimilating different N sources can alter redox homeostasis and antioxidant systems in different cellular compartments, including the mitochondria and the cell wall. Conversely, changes in mitochondrial redox systems can affect plant responses to N. This review explores the integration between N assimilation, mitochondrial redox metabolism, and apoplast metabolism.

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants: A NOVEL ENZYME IN PHOSPHATIDYLCHOLINE SYNTHESIS.

Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).

The evolution of substrate specificity-associated residues and Ca(2+) -binding motifs in EF-hand-containing type II NAD(P)H dehydrogenases.

Most eukaryotic organisms, except some animal clades, have mitochondrial alternative electron transport enzymes that allow respiration to bypass the energy coupling in oxidative phosphorylation. The energy bypass enzymes in plants include the external type II NAD(P)H dehydrogenases (DHs) of the NDB family, which are characterized by an EF-hand domain for Ca(2+) binding. Here we investigate these plant enzymes by combining molecular modeling with evolutionary analysis. Molecular modeling of the Arabidopsis thaliana AtNDB1 with the yeast ScNDI1 as template revealed distinct similarities in the core catalytic parts, and highlighted the interaction between the pyridine nucleotide and residues correlating with NAD(P)H substrate specificity. The EF-hand domain of AtNDB1 has no counterpart in ScNDI1, and was instead modeled with Ca(2+) -binding signal transducer proteins. Combined models displayed a proximity of the AtNDB1 EF-hand domain to the substrate entrance side of the catalytic part. Evolutionary analysis of the eukaryotic NDB-type proteins revealed ancient and recent reversions between the motif observed in proteins specific for NADH (acidic type) and NADPH (non-acidic type), and that the clade of enzymes with acidic motifs in angiosperms derives from non-acidic-motif NDB-type proteins present in basal plants, fungi and protists. The results suggest that Ca(2+) -dependent external NADPH oxidation is an ancient process, indicating that it has a fundamental importance for eukaryotic cellular redox metabolism. In contrast, the external NADH DHs in plants are products of a recent expansion, mirroring the expansion of the alternative oxidase family.

The Ca2+-Regulation of the Mitochondrial External NADPH Dehydrogenase in Plants Is Controlled by Cytosolic pH.

NADPH is a key reductant carrier that maintains internal redox and antioxidant status, and that links biosynthetic, catabolic and signalling pathways. Plants have a mitochondrial external NADPH oxidation pathway, which depends on Ca2+ and pH in vitro, but concentrations of Ca2+ needed are not known. We have determined the K0.5(Ca2+) of the external NADPH dehydrogenase from Solanum tuberosum mitochondria and membranes of E. coli expressing Arabidopsis thaliana NDB1 over the physiological pH range using O2 and decylubiquinone as electron acceptors. The K0.5(Ca2+) of NADPH oxidation was generally higher than for NADH oxidation, and unlike the latter, it depended on pH. At pH 7.5, K0.5(Ca2+) for NADPH oxidation was high (≈100 µM), yet 20-fold lower K0.5(Ca2+) values were determined at pH 6.8. Lower K0.5(Ca2+) values were observed with decylubiquinone than with O2 as terminal electron acceptor. NADPH oxidation responded to changes in Ca2+ concentrations more rapidly than NADH oxidation did. Thus, cytosolic acidification is an important activator of external NADPH oxidation, by decreasing the Ca2+-requirements for NDB1. The results are discussed in relation to the present knowledge on how whole cell NADPH redox homeostasis is affected in plants modified for the NDB1 gene.

Studies on the expression of linalool synthase using a promoter-ß-glucuronidase fusion in transgenic Artemisia annua.

Artemisinin, an antimalarial endoperoxide sesquiterpene, is synthesized in glandular trichomes of Artemisia annua L. A number of other enzymes of terpene metabolism utilize intermediates of artemisinin biosynthesis, such as isopentenyl and farnesyl diphosphate, and may thereby influence the yield of artemisinin. In order to study the expression of such enzymes, we have cloned the promoter regions of some enzymes and fused them to ß-glucuronidase (GUS). In this study, we have investigated the expression of the monoterpene synthase linalool synthase (LIS) using transgenic A. annua carrying the GUS gene under the control of the LIS promoter. The 652bp promoter region was cloned by the genome walker method. A number of putative cis-acting elements were predicted indicating that the LIS is driven by a complex regulation mechanism. Transgenic plants carrying the promoter-GUS fusion showed specific expression of GUS in T-shaped trichomes (TSTs) but not in glandular secretory trichomes, which is the site for artemisinin biosynthesis. GUS expression was observed at late stage of flower development in styles of florets and in TSTs and guard cells of basal bracts. GUS expression after wounding showed that LIS is involved in plant responsiveness to wounding. Furthermore, the LIS promoter responded to methyl jasmonate (MeJA). These results indicate that the promoter carries a number of cis-acting regulatory elements involved in the tissue-specific expression of LIS and in the response of the plant to wounding and MeJA treatment. Southern blot analysis indicated that the GUS gene was integrated in the A. annua genome as single or multi copies in different transgenic lines. Promoter activity analysis by qPCR showed that both the wild-type and the recombinant promoter are active in the aerial parts of the plant while only the recombinant promoter was active in roots. Due to the expression in TSTs but not in glandular trichomes, it may be concluded that LIS expression will most likely have little or no effect on artemisinin production.