Biology subjects

Fernandes, P. A.

Publications and source records attributed to Fernandes, P. A..

4 recordsLinked to original sources

bioRxiv2026

Expanding the Enzymatic Landscape for Polyurethane Degradation of Novel Bacterial Urethanases

Polyurethanes (PURs) represent a significant challenge in plastic waste management due to their chemical resilience and limited recycling options. In this study, we report the identification and characterization of five novel bacterial urethanases, expanding the enzymatic repertoire for targeted PUR depolymerization. These enzymes demonstrated carbamate-cleaving activity optimally under alkaline conditions, maintaining stability across a pH range of 7 to 10 and varying thermal and solvent tolerances. Two candidate enzymes, u17 u15 collectively exhibited high activity, catalytic efficiency, and thermostability, establishing a strong foundation for further optimization. Among them, u15 emerged as particularly notable for its catalytic efficiency on the carbamate model substrate di-urethane ethylene methylenedianiline, DUE-MDA, with a kcat/KM of 51.8 {+/-} 0.1 (s-1mM-1). and this motivated its selection for detailed structural analysis. High-resolution crystallography of u15 revealed key active-site architecture, including the conserved amidase signature catalytic triad and flexible loop regions that influence substrate binding and specificity. Molecular docking and molecular dynamics simulations further elucidated substrate binding determinants of u15 during urethane bond hydrolysis. Docking of DUE-MDA revealed two distinct substrate orientations (Pose A and Pose B) differing in the positioning of the carbamate group relative to Ser177. Pose A was more stable and catalytically competent, maintaining the substrate within the oxyanion hole and sustaining optimal geometry for nucleophilic attack by Ser177. Comparable behavior was observed for the partially hydrolyzed intermediate mono-urethane ethylene methylenedianiline, MUE-MDA, indicating a conserved binding mode across substrates. To further assess enzymatic performance on a realistic industrial material, the panel was then tested on a generic flexible foam substrate derived from 2,4- and 2,6-toluene diisocyanate (TDA), where u15 and u17 emerged as the most active candidates. Collectively, we benchmark the structural framework presented by enzymes in the amidase signature family as a strong foundation for further optimization aiming at advancing sustainable and scalable biocatalytic recycling of polyurethanes.

biochemistry↗

bioRxiv2024

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the {beta}-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. Intriguingly, the KS protomers within the dimer exhibit positive cooperativity, investigated by mutational studies and acyl-carrier analysis, which likely serves the regulation of biosynthesis. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

biochemistry↗

bioRxiv2024

Snake venom-inspired novel peptides protect Caenorhabditis elegans against paraquat induced Parkinson's pathology

The in vivo protective mechanisms of two low molecular mass ([~]1.4 kDa) novel custom peptides (CPs) against paraquat (PT)-induced neurodegenerative dysfunction in the Caenorhabditis elegans model were deciphered. CPs prevent the PT binding to the nerve ring adjacent to the pharynx in C. elegans (N2 strain) by stable and high-affinity binding to the tyrosine-protein kinase receptor CAM-1, resulting in significant inhibition of PT-induced toxicity by reducing enhanced reactive oxygen species production, mitochondrial membrane depolarization, and chemosensory dysfunction. The CPs inhibited PT-induced dopaminergic (DAergic) neuron degeneration and alpha-synuclein aggregation, the hallmarks of Parkinsons Disease, in transgenic BZ555 and NL5901 strains of C. elegans. The transcriptomic, functional proteomics, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses show that CPs prevented the increased expression of the genes involved in the skn-1 downstream pathway, thereby restoring PT-mediated oxidative stress, apoptosis, and neuronal damage in C. elegans. The CPs ability to repair PT-induced damage was demonstrated by a network of gene expression profiles illustrating the molecular relationships between the regulatory proteins. Further, CPs (10 mg/kg, parental route) did not show toxicity or induce inflammatory mediators in the mouse model.

neuroscience↗

bioRxiv2019

Pan-cancer single cell RNA-seq uncovers recurring programs of cellular heterogeneity

Cultured cell lines are the workhorse of cancer research, but it is unclear to what extent they recapitulate the cellular heterogeneity observed among malignant cells in tumors, given the absence of a native tumor microenvironment. Here, we used multiplexed single cell RNA-seq to profile ~200 cancer cell lines. We uncovered expression programs that are recurrently heterogeneous within many cancer cell lines and are largely independent of observed genetic diversity. These programs of heterogeneity are associated with diverse biological processes, including cell cycle, senescence, stress and interferon responses, epithelial-to-mesenchymal transition, and protein maturation and degradation. Notably, some of these recurrent programs recapitulate those seen in human tumors, suggesting a prominent role of intrinsic plasticity in generating intra-tumoral heterogeneity. Moreover, the data allowed us to prioritize specific cell lines as model systems of cellular plasticity. We used two such models to demonstrate the dynamics, regulation and drug sensitivities associated with a cancer senescence program also observed in human tumors. Our work describes the landscape of cellular heterogeneity in diverse cancer cell lines, and identifies recurrent patterns of expression heterogeneity that are shared between tumors and specific cell lines and can thus be further explored in follow up studies.

cancer biology↗