Biology subjects

Prentice, E.

Publications and source records attributed to Prentice, E..

4 recordsLinked to original sources

bioRxiv2025

Characterisation of O-acetylserine sulfhyrdrylase (CysK) enzymes from bacteria lacking a sulfate reduction pathway

Sulfur metabolism plays an important role in bacterial pathogenesis. Elucidation of differences in sulfur metabolism across bacterial pathogens furthers our understanding of host survival and offers opportunities to disrupt these pathways for new therapies. Withing bacteria sulfur metabolism converges at the synthesis of L-cysteine. One of the key mechanisms of obtaining sulfur for the synthesis of L-cysteine is the successive reduction of sulfate to sulfide via the sulfate reduction pathway. Accordingly, L-cysteine biosynthesis is a critical metabolic pathway for bacterial survival, particularly in pathogenic species such as Neisseria gonorrhoeae and Staphylococcus aureus, which lack the sulfate reduction pathway. O-acetylserine sulfhydrylase catalyses the second step of the two-step synthesis reaction, condensing sulfide or thiosulfate (in the case of OASS-A/CysK or OASS-B/CysM respectively) with O-acetylserine to synthesize cysteine. Here we investigate the enzymatic properties and functional characterization of O-acetylserine sulfhydrylase, from N. gonorrhoeae and S. aureus, with a focus on substrate specificity, kinetic parameters, and cysteine synthase complex (CSC) formation. Using small angle X-ray scattering and kinetic assays we demonstrate that both N. gonorrhoeae and S. aureus CysK enzymes utilise only sodium sulfide for the synthesis of cysteine, despite the lack of a sulfate reduction pathway (to generate sulfide) in these organisms. Both enzymes demonstrate a higher affinity for O-acetylserine (OAS) compared to sodium sulfide (Na2S). We also show that the two cysteine synthesis enzymes, CysE and CysK that traditionally form the cysteine synthase complex do not form a complex in N. gonorrhoeae. These findings highlight the functional divergence in sulfur metabolism strategies among bacteria lacking sulfate reduction and provide deeper insights into the adaptive mechanisms of N. gonorrhoeae and S. aureus in sulfur flux.

biochemistry↗

bioRxiv2024

Identification of novel inhibitors targeting serine acetyltransferase from Neisseria gonorrhoeae

Neisseria gonorrhoeae is an obligate human pathogen and the etiological agent of the sexually transmitted infection, gonorrhoea. The rapid emergence of extensively antimicrobial-resistant strains, including those resistant to all frontline antibiotics, has led to N. gonorrhoeae being labelled a priority pathogen by the World Health Organization, highlighting the need for new antimicrobial treatments. Given its absence in humans, targeting de novo cysteine biosynthesis has been identified as a promising avenue for developing new antimicrobials against drug-resistant bacteria. The biosynthesis of cysteine is catalyzed by two enzymes; serine acetyltransferase (SAT/CysE) which catalyzes the first step and O-acetylserine sulfhydrylase (OASS/CysK) that catalyzes the second step incorporating sulfur to form L-cysteine. CysE is reported to be essential for bacterial survival in several bacterial pathogens including N. gonorrhoeae. Here, we have conducted virtual inhibitor screening of commercially available compound libraries against SAT from N. gonorrhoeae (NgSAT). We have identified a hit compound with an IC50 of 13.9 {micro}M and analyzed its interactions with the enzymes active site. This provides a platform for the identification and development of novel SAT inhibitors to combat drug-resistant bacterial pathogens.

biochemistry↗

bioRxiv2024

Modified histone peptides uniquely tune the material properties of HP1α condensates.

Biomolecular condensates have emerged as a powerful new paradigm in cell biology with broad implications to human health and disease, particularly in the nucleus where phase separation is thought to underly elements of chromatin organization and regulation. Specifically, it has been recently reported that phase separation of heterochromatin protein 1alpha (HP1) with DNA contributes to the formation of condensed chromatin states. HP1 localization to heterochromatic regions is mediated by its binding to specific repressive marks on the tail of histone H3, such as trimethylated lysine 9 on histone H3 (H3K9me3). However, whether epigenetic marks play an active role in modulating the material properties of HP1 and dictating emergent functions of its condensates, remains only partially understood. Here, we leverage a reductionist system, comprised of modified and unmodified histone H3 peptides, HP1 and DNA to examine the contribution of specific epigenetic marks to phase behavior of HP1. We show that the presence of histone peptides bearing the repressive H3K9me3 is compatible with HP1 condensates, while peptides containing unmodified residues or bearing the transcriptional activation mark H3K4me3 are incompatible with HP1 phase separation. In addition, inspired by the decreased ratio of nuclear H3K9me3 to HP1 detected in cells exposed to uniaxial strain, using fluorescence microscopy and rheological approaches we demonstrate that H3K9me3 histone peptides modulate the dynamics and network properties of HP1 condensates in a concentration dependent manner. These data suggest that HP1-DNA condensates are viscoelastic materials, whose properties may provide an explanation for the dynamic behavior of heterochromatin in cells in response to mechanostimulation. Statement of significanceThe organization of genomic information in eukaryotic cells necessitates compartmentalization into functional domains allowing for the expression of cell identity-specific genes, while repressing genes related to alternative fates. Heterochromatin hosts these transcriptionally silent regions of the genome - which ensure the stability of cell identity -and is characterized by repressive histone marks (H3K9m3) and other specialized proteins (HP1a), recently shown to phase separate with DNA. We show that HP1a forms condensates with DNA which persist in the presence of H3K9me3 peptides. The viscoelastic nature of these condensates depend on H3K9me3:HP1 ratios, which are modulated by mechanical strain in cells. Thus, phase separation may explain the dynamic behavior of heterochromatin in cells, in response to mechanostimulation.

biophysics↗

bioRxiv2021

Chemical mapping exposes the importance of active site interactions in governing the temperature dependence of enzyme turnover

Uncovering the role of global protein dynamics in enzyme turnover is needed to fully understand enzyme catalysis. Recently, we have demonstrated that the heat capacity of catalysis, {Delta}CP{ddagger}, can reveal links between the protein free energy landscape, global protein dynamics and enzyme turnover, suggesting that subtle changes molecular interactions at the active site can affect long range protein dynamics and link to enzyme temperature activity. Here we use a model promiscuous enzyme (Glucose dehydrogenase from Sulfolobus Solfataricus) to chemically map how individual substrate interactions affect the temperature dependence of enzyme activity and the network of motions throughout the protein. Utilizing a combination of kinetics, REES spectroscopy and computational simulation we explore the complex relationship between enzyme-substrate interactions and the global dynamics of the protein. We find that changes in {Delta}CP{ddagger} and protein dynamics can be mapped to specific substrate-enzyme interactions. Our study reveals how subtle changes in substrate binding affect global changes in motion and flexibility extending throughout the protein.

biochemistry↗