Biology subjects

Walsh, B. J. C.

Publications and source records attributed to Walsh, B. J. C..

3 recordsLinked to original sources

bioRxiv2026

Self-S-sulfonation in a bacterial persulfide dioxygenase mediates thiol persulfide detoxification

A ubiquitous class of non-heme Fe(II) enzymes, the persulfide dioxygenases (PDOs), provide protection against hydrogen sulfide (H2S) poisoning. The PDO in humans is a single-domain enzyme, while bacterial PDOs, such as CstB of Staphylococcus aureus, are often fused to a sulfurtransferase (rhodanese) module. Canonical PDOs cleave the S-S bond of glutathione persulfide (GSSH) to produce GSH and sulfite (SO32-). In contrast, CstB, via an unknown mechanism, converts two RSSH to thiosulfate (S2O32-) without the release of sulfite. Six crystallographic structures of S. aureus CstB reveal that a Cys-Gly sequence (C201-G202) in a CstB-unique dynamic loop functions as a glutathione mimic, occupying one face of the hemifacial octahedral Fe(II) coordination site. We establish that CstB self-S-sulfonates C201 in a thiol persulfide, Fe(II) and O2-dependent manner, which is then shuttled to a persulfidated C408 in the rhodanese domain {approx}27 [A] away via electrostatic steering to generate thiosulfate as the sole oxidation product. Both C201A and C408A CstBs are inactive in O2-consumption. Self-S-sulfonation ensures rapid clearance of diverse reactive sulfur species under conditions where these species accumulate, permitting S. aureus to harness their cytoprotective effects while avoiding cellular toxicity.

biochemistry↗

bioRxiv2025

DISCOVERY AND BIOCHEMICAL CHARACTERIZATION OF A FUNGAL ICE NUCLEATION PROTEIN FROM PODILA CLONOCYSTIS

Biological ice nucleation plays a pivotal role in atmospheric processes, yet the molecular basis of fungal ice nucleation remains poorly understood compared to bacterial systems. Here, we report the biochemical characterization of an ice nucleation protein (PcINP) from a soil-dwelling fungus Podila clonocystis, not previously reported to produce ice nuclei. Using sequence similarity network analysis, we identified PcINP as a putative fungal homolog of bacterial ice nucleation proteins and confirmed its function through recombinant expression in Escherichia coli. We probe the function of PcINP structure through domain truncations and demonstrate that a poorly structured N-terminal region is not necessary for ice nucleation activity and can be functionally replaced with an expression enhancing SUMO fusion tag. Finally, we observe both monomeric and aggregated PcINP in E. coli lysates using SEC-MALS but are unable to distinguish their ice nucleation activity pointing to an unknown in vitro aggregation mechanism. Our findings establish PcINP within the emerging class fungal ice nucleation protein with distinct structural features and high stability, expanding the known diversity of biological ice nucleators and highlighting their potential for environmental and biotechnological applications.

biochemistry↗

bioRxiv2020

Structural determinants of persulfide-sensing specificity in a dithiol-based transcriptional regulator

Cysteine thiol-based transcriptional regulators orchestrate coordinated regulation of redox homeostasis and other cellular processes by "sensing" or detecting a specific redox-active molecule, which in turn activates the transcription of a specific detoxification pathway. The extent to which these sensors are truly specific in cells for a singular class of reactive small molecule stressors, e.g., reactive oxygen or sulfur species, is largely unknown. Here we report novel structural and mechanistic insights into a thiol-based transcriptional repressor SqrR, that reacts exclusively with organic and inorganic oxidized sulfur species, e.g., persulfides, to yield a unique tetrasulfide bridge that allosterically inhibits DNA operator-promoter binding. Evaluation of five crystallographic structures of SqrR in various derivatized states, coupled with the results of a mass spectrometry-based kinetic profiling strategy, suggest that persulfide selectivity is determined by structural frustration of the disulfide form. This energetic roadblock effectively decreases the reactivity toward major oxidants to kinetically favor formation of the tetrasulfide product. These findings lead to the identification of an uncharacterized repressor from the increasingly antibiotic-resistant bacterial pathogen, Acinetobacter baumannii, as a persulfide sensor, illustrating the predictive power of this work and potential applications to bacterial infectious disease.

biochemistry↗