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Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation
The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes.
Biochemical and structural basis for differential inhibitor sensitivity of EGFR with distinct exon 1
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Biochemical implementation of acceleration sensing and PIDA control
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Author Correction: A map of the rubisco biochemical landscape
Correction to: Naturehttps://doi.org/10.1038/s41586-024-08455-0 Published online 22 January 2025. In the version of the article initially published, the affiliations of Hana A. Chang (Department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA, USA) and Ron Milo (Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel) were incorrect and have now been amended in the HTML and PDF versions of the article.
Metabolic Profiling Reveals Biochemical Pathways Responsible for Eelgrass Response to Elevated CO2 and Temperature
Abstract As CO 2 levels in Earth’s atmosphere and oceans steadily rise, varying organismal responses may produce ecological losers and winners. Increased ocean CO 2 can enhance seagrass productivity and thermal tolerance, providing some compensation for climate warming. However, the metabolic shifts driving the positive response to elevated CO 2 by these important ecosystem engineers remain unknown. We analyzed whole-plant performance and metabolic profiles of two geographically distinct eelgrass ( Zostera marina L.) populations in response to CO 2 enrichment. In addition to enhancing overall plant size, growth and survival, CO 2 enrichment increased the abundance of Calvin Cycle and nitrogen assimilation metabolites while suppressing the abundance of stress-related metabolites. Overall metabolome differences between populations suggest that some eelgrass phenotypes may be better suited than others to cope with an increasingly hot and sour sea. Our results suggest that seagrass populations will respond variably, but overall positively, to increasing CO 2 concentrations, generating negative feedbacks to climate change.
In-depth quantitative proteomic characterization of organotypic hippocampal slice culture reveals sex-specific differences in biochemical pathways
Sex differences in the brain of mammals range from neuroarchitecture through cognition to cellular metabolism. The hippocampus, a structure mostly associated with learning and memory, presents high vulnerability to neurodegeneration and aging. Therefore, we explored basal sex-related differences in the proteome of organotypic hippocampal slice culture, a major in vitro model for studying the cellular and molecular mechanisms related to neurodegenerative disorders. Results suggest a greater prevalence of astrocytic metabolism in females and significant neuronal metabolism in males. The preference for glucose use in glycolysis, pentose phosphate pathway and glycogen metabolism in females and high abundance of mitochondrial respiration subunits in males support this idea. An overall upregulation of lipid metabolism was observed in females. Upregulation of proteins responsible for neuronal glutamate and GABA synthesis, along with synaptic associated proteins, were observed in males. In general, the significant spectrum of pathways known to predominate in neurons or astrocytes, together with the well-known neuronal and glial markers observed, revealed sex-specific metabolic differences in the hippocampus. TEM qualitative analysis might indicate a greater presence of mitochondria at CA1 synapses in females. These findings are crucial to a better understanding of how sex chromosomes can influence the physiology of cultured hippocampal slices and allow us to gain insights into distinct responses of males and females on neurological diseases that present a sex-biased incidence.
Omics analyses and biochemical study of Phlebiopsis gigantea elucidate its degradation strategy of wood extractives
Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi.
Molecular and biochemical characterization of rice developed through conventional integration of nDart1-0 transposon gene
Mutations, the genetic variations in genomic sequences, play an important role in molecular biology and biotechnology. During DNA replication or meiosis, one of the mutations is transposons or jumping genes. An indigenous transposon nDart1-0 was successfully introduced into local indica cultivar Basmati-370 from transposon-tagged line viz., GR-7895 (japonica genotype) through conventional breeding technique, successive backcrossing. Plants from segregating populationsshowed variegated phenotypes were tagged as BM-37 mutants. Blast analysis of the sequence data revealed that the GTP-binding protein, located on the BAC clone OJ1781_H11 of chromosome 5, contained an insertion of DNA transposon nDart1-0. The nDart1-0 has “A” at position 254 bp, whereas nDart1 homologs have “G”, which efficiently distinguishes nDart1-0 from its homologs. The histological analysis revealed that the chloroplast of mesophyll cells in BM-37 was disrupted with reduction in size of starch granules and higher number of osmophillic plastoglobuli, which resulted in decreased chlorophyll contents and carotenoids, gas exchange parameters (Pn, g, E, Ci), and reduced expression level of genes associated with chlorophyll biosynthesis, photosynthesis and chloroplast development. Along with the rise of GTP protein, the salicylic acid (SA) and gibberellic acid (GA) and antioxidant contents(SOD) and MDA levels significantly enhanced, while, the cytokinins (CK), ascorbate peroxidase (APX), catalase (CAT), total flavanoid contents (TFC) and total phenolic contents (TPC) significantly reduced in BM-37 mutant plants as compared with WT plants. These results support the notion that GTP-binding proteins influence the process underlying chloroplast formation. Therefore, it is anticipated that to combat biotic or abiotic stress conditions, the nDart1-0 tagged mutant (BM-37) of Basmati-370 would be beneficial.
Identifying Bayesian optimal experiments for uncertain biochemical pathway models
Abstract Pharmacodynamic (PD) models are mathematical models of cellular reaction networks that include drug mechanisms of action. These models are useful for studying predictive therapeutic outcomes of novel drug therapies in silico. However, PD models are known to possess significant uncertainty with respect to constituent parameter data, leading to uncertainty in the model predictions. Furthermore, experimental data to calibrate these models is often limited or unavailable for novel pathways. In this study, we present a Bayesian optimal experimental design approach for improving PD model prediction accuracy. We then apply our method using simulated experimental data to account for uncertainty in hypothetical laboratory measurements. This leads to a probabilistic prediction of drug performance and a quantitative measure of which prospective laboratory experiment will optimally reduce prediction uncertainty in the PD model. The methods proposed here provide a way forward for uncertainty quantification and guided experimental design for models of novel biological pathways.
Insights on autophagosome–lysosome tethering from structural and biochemical characterization of human autophagy factor EPG5
Pivotal to the maintenance of cellular homeostasis, macroautophagy (hereafter autophagy) is an evolutionarily conserved degradation system that involves sequestration of cytoplasmic material into the double-membrane autophagosome and targeting of this transport vesicle to the lysosome/late endosome for degradation. EPG5 is a large-sized metazoan protein proposed to serve as a tethering factor to enforce autophagosome–lysosome/late endosome fusion specificity, and its deficiency causes a severe multisystem disorder known as Vici syndrome. Here, we show that human EPG5 (hEPG5) adopts an extended “shepherd’s staff” architecture. We find that hEPG5 binds preferentially to members of the GABARAP subfamily of human ATG8 proteins critical to autophagosome–lysosome fusion. The hEPG5–GABARAPs interaction, which is mediated by tandem LIR motifs that exhibit differential affinities, is required for hEPG5 recruitment to mitochondria during PINK1/Parkin-dependent mitophagy. Lastly, we find that the Vici syndrome mutation Gln336Arg does not affect the hEPG5’s overall stability nor its ability to engage in interaction with the GABARAPs. Collectively, results from our studies reveal new insights into how hEPG5 recognizes mature autophagosome and establish a platform for examining the molecular effects of Vici syndrome disease mutations on hEPG5.
Biochemical and structural characterization of Haemophilus influenzae nitroreductase in metabolizing
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The formulation of chemical potentials and free energy changes in biochemical reactions
Critical evaluations of IUPAC recommendations for (1) pH transforms and (2) the 1994 Alberty and recent BBR methods for calculating reaction free energies shows that pH transforms are unnecessary and the two reaction free energies are not the same.
Structural and biochemical characterisation of Co 2+ -binding sites on serum albumins and their interplay with fatty acids
Serum albumin–Co 2+ interactions are of clinical importance. They play a role in mediating the physiological effects associated with cobalt toxicity and are central to the albumin cobalt binding (ACB) assay for diagnosis of myocardial ischemia. To further understand these processes, a deeper understanding of albumin–Co 2+ interactions is required. Here, we present the first crystallographic structures of human serum albumin (HSA; three structures) and equine serum albumin (ESA; one structure) in complex with Co 2+ . Amongst a total of sixteen sites bearing a cobalt ion across the structures, two locations were prominent, and they relate to metal-binding sites A and B. Site-directed mutagenesis and isothermal titration calorimetry (ITC) were employed to characterise sites on HSA. The results indicate that His9 and His67 contribute to the primary (putatively corresponding to site B) and secondary Co 2+ -binding sites (site A), respectively. The presence of additional multiple weak-affinity Co 2+ binding sites on HSA was also supported by ITC studies. Furthermore, addition of 5 molar equivalents of the non-esterified fatty acid palmitate (C16:0) reduced the Co 2+ -binding affinity at both sites A and B. The presence of bound myristate (C14:0) in the HSA crystal structures provided insight into the fatty acid-mediated structural changes that diminish the affinity of the protein toward Co 2+ . Together, these data provide further support for the idea that ischemia-modified albumin corresponds to albumin with excessive fatty-acid loading. Collectively, our findings provide a comprehensive understanding of the molecular underpinnings governing Co 2+ binding to serum albumin.
Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism
Significance More than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.