Biology subjects

Bomble, Y. J.

Publications and source records attributed to Bomble, Y. J..

6 recordsLinked to original sources

bioRxiv2024

Decoding substrate specificity determining factors in glycosyltransferase-B enzymes - Insights from machine learning models

Substrate specificity is an essential characteristic of any enzymes function and an understanding of the factors that determine this specificity is crucial for enzyme engineering. Unlike the structure of an enzyme which is directly impacted by its sequence, substrate specificity as an enzyme attribute involves a rather indirect relationship with sequence as it also depends on structural aspects that dictate substrate accessibility and active site dynamics. In this study, we explore the performance of classifier-based machine learning models trained on curated sequence and structural data for a class of glycosyltransferases (GTs), namely GT-Bs, to understand their substrate specificity determining factors. GTs enable the transfer of sugar moieties to other biomolecules such as oligosaccharides or proteins and are found in all kingdoms of life. In plants, GTs participate in the biosynthesis of plant cell wall biopolymers (eg: hemicelluloses and pectins) and are an integral part of the enzymatic machinery that enables the storage of carbon and energy as plant biomass. To elucidate the substrate specificity of uncharacterized GT-Bs, we constructed multi-label machine learning models (Support Vector Classifier, K-Nearest Neighbors, Gaussian Naive-Bayes, Random Forest) that incorporate both sequence and structural features. These models achieve good predictive accuracies on test datasets. However, despite our use of structural information, we highlight that there is further scope for improvement in training these models to draw interpretable relationships between sequence, structure and substrate specificity determining motifs in GT-Bs.

bioinformatics↗

bioRxiv2024

Structural characterization and dynamics of AdhE ultrastructures from Clostridium thermocellum: A containment strategy for toxic intermediates?

Clostridium thermocellum, a cellulolytic thermophilic anaerobe, is considered by many to be a prime candidate for the realization of consolidated bioprocessing (CBP) and is known as an industry standard for biofuel production. C. thermocellum is among the best biomass degraders identified to date in nature and produces ethanol as one of its main products. Many studies have helped increase ethanol titers in this microbe, however ethanol production using C. thermocellum is still not economically viable. Therefore, a better understanding of its ethanol synthesis pathway is required. The main pathway for ethanol production in C. thermocellum involves the bifunctional aldehyde-alcohol dehydrogenase (AdhE). To better understand the function of the C. thermocellum AdhE, we used cryo-electron microscopy (cryo-EM) to obtain a 3.28 [A] structure of the AdhE complex. This high-resolution structure, in combination with molecular dynamics simulations, provides insight into the substrate channeling of the toxic intermediate acetaldehyde, indicates the potential role of C. thermocellum AdhE to regulate activity and cofactor pools, and establishes a basis for future engineering studies. The containment strategy found in this enzyme offers a template that could be replicated in other systems where toxic intermediates need to be sequestered to increase the production of valuable biochemicals.

biophysics↗

bioRxiv2023

Engineering and characterization of carbohydrate-binding modules to enable real-time imaging of cellulose fibrils biosynthesis in plant protoplasts

O_LICarbohydrate binding modules (CBMs) are non-catalytic domains associated with cell wall degrading carbohydrate-active enzymes (CAZymes) that are often present in nature tethered to distinct catalytic domains (CD). Fluorescently labeled CBMs have been also used to visualize the presence of specific polysaccharides present in the cell wall of plant cells and tissues. C_LIO_LIPrevious studies have provided a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of optimal CBM designs for recognizing specific plant cell wall glycans. Furthermore, CBMs also have not been used to study cell wall regeneration in plant protoplasts. C_LIO_LIHere, we examine the dynamic interactions of engineered type-A CBMs (from families 3a and 64) with crystalline cellulose-I and phosphoric acid swollen cellulose (PASC). We generated tandem CBM designs to determine their binding parameters and reversibility towards cellulose-I using equilibrium binding assays. Kinetic parameters - adsorption (kon) and desorption (koff) rate constants-for CBMs towards nanocrystalline cellulose were determined using quartz crystal microbalance with dissipation (QCM-D). Our results indicate that tandem CBM3a exhibits a five-fold increased adsorption rate to cellulose compared to single CBM3a, making tandem CBM3a suitable for live-cell imaging applications. We next used engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using wide-field fluorescence and confocal laser scanning microscopy (CLSM). C_LIO_LIIn summary, tandem CBMs offer a novel polysaccharide labeling probe for real-time visualization of growing cellulose chains in living Arabidopsis protoplasts. C_LI

plant biology↗

bioRxiv2020

Structure of the Xylan O-Acetyltransferase AtXOAT1 Reveals Molecular Insight into Polysaccharide Acetylation in Plants

Acetylation of biomolecules is gaining increased attention due to both the abundance and importance of this modification across all kingdoms of life. Xylans are a major component of plant cell walls and are the third most abundant biopolymer in Nature. O-Acetyl moieties are the dominant backbone substituents of glucuronoxylan in dicots and play a major role in the polymer-polymer interactions that are crucial for proper wall architecture and normal plant development. Here, we describe the biochemical, structural, and mechanistic characterization of Arabidopsis thaliana xylan O-acetyltransferase 1 (AtXOAT1), a member of the plant-specific Trichome Birefrigence Like (TBL) family that catalyzes the 2-O-acetylation of xylan. A multipronged approach involving X-ray crystallography, biochemical analyses, mutagenesis, and molecular simulations show that XOAT1 catalyzes xylan acetylation through formation of an acyl-enzyme intermediate by a double displacement bi-bi mechanism involving a Ser-His-Asp catalytic triad and unconventionally employs an arginine residue in formation of an oxyanion hole.

biochemistry↗

bioRxiv2018

Bayesian inference of metabolic kinetics from genome-scale multiomics data

Modern biological tools generate a wealth of data on metabolite and protein concentrations that can be used to help inform new strain designs. However, integrating these data sources to generate predictions of steady-state metabolism typically requires a kinetic description of the enzymatic reactions that occur within a cell. Parameterizing these kinetic models from biological data can be computationally difficult, especially as the amount of data increases. Robust methods must also be able to quantify the uncertainty in model parameters as a function of the available data, which can be particularly computationally intensive. The field of Bayesian inference offers a wide range of methods for estimating distributions in parameter uncertainty. However, these techniques are poorly suited to kinetic metabolic modeling due to the complex kinetic rate laws typically employed and the resulting dynamic system that must be solved. In this paper, we employ linear-logarithmic kinetics to simplify the calculation of steady-state flux distributions and enable efficient sampling and variational inference methods. We demonstrate that detailed information on the posterior distribution of kinetic model parameters can be obtained efficiently at a variety of different problem scales, including large-scale kinetic models trained on multiomics datasets. These results allow modern Bayesian machine learning tools to be leveraged in understanding biological data and developing new, efficient strain designs.

systems biology↗

bioRxiv2018

Comparative Biochemical and Structural Analysis of Novel Cellulose Binding Proteins (Tapirins) from Extremely Thermophilic Caldicellulosiruptor Species

Genomes of extremely thermophilic Caldicellulosiruptor species encode novel cellulose binding proteins, t[a]pirins, located proximate to the type IV pilus locus. Previously, the C-terminal domain of a t[a]pirin (Calkro_0844) from Caldicellulosiruptor kronotskyensis was shown to be structurally unique and have a cellulose binding affinity akin to family 3 carbohydrate binding modules (CBM3). Here, full-length and C-terminal versions of t[a]pirins from Caldicellulosiruptor bescii (Athe_1870), Caldicellulosiruptor hydrothermalis (Calhy_0908), Caldicellulosiruptor kristjanssonii (Calkr_0826), and Caldicellulosiruptor naganoensis (NA10_0869) were produced recombinantly in Escherichia coli and compared to Calkro_0844. All five t[a]pirins bound to microcrystalline cellulose, switchgrass, poplar, filter paper, but not to xylan. Densitometry analysis of bound protein fractions visualized by SDS-PAGE revealed that Calhy_0908 and Calkr_0826 (from weakly cellulolytic species) associated with the cellulose substrates to a greater extent than Athe_1870, Calkro_0844 and NA10_0869 (from strongly cellulolytic species), perhaps to associate closely with biomass to capture glucans released from lignocellulose by cellulases produced in Caldicellulosiruptor communities. Three-dimensional structures of the C-terminal binding regions of Calhy_0908 and Calkr_0826 were closely related to Calkro_0844, despite the fact that their amino acid sequence identities compared to Calkro_0844 were only 16% and 36%, respectively. Unlike the parent strain, C. bescii mutants lacking the t[a]pirin genes did not bind to cellulose following short-term incubation, reinforcing the significance of these proteins in cell association with plant biomass. Given the scarcity of carbohydrates in neutral terrestrial hot springs, t[a]pirins likely help cells scavenge carbohydrates from lignocellulose to support growth and survival of Caldicellulosiruptor species.\n\nImportanceMechanisms by which microorganisms attach to and degrade lignocellulose are important to understand if effective approaches for conversion of plant biomass into fuels and chemicals are to be developed. Caldicellulosiruptor species grow on carbohydrates from lignocellulose at elevated temperatures and have biotechnological significance for that reason. Novel cellulose binding proteins, called t[a]pirins, are involved in the way Caldicellulosiruptor species interact with microcrystalline cellulose and here additional information about the diversity of these proteins across the genus is provided, including three dimensional structural comparisons.

microbiology↗