Biology subjects

Mazgaj, R.

Publications and source records attributed to Mazgaj, R..

5 recordsLinked to original sources

bioRxiv2026

An engineered biofactory for efficient production of diverse recombinant superoxide dismutase isozymes loaded with specific metal ions for biochemical characterisation

BackgroundBiochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. ResultsGenomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. ConclusionsThe E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

microbiology↗

bioRxiv2025

A closely related pair of superoxide dismutase isozymes from Staphylococcus aureus show distinct stabilities and proton-exchange dynamics

Changes in biochemical properties, caused by iterative mutations in amino acid sequence, underlie the alterations in protein function over time that underpin the evolutionary process. An example is the switching of an enzymes reliance from one essential metal to an alternative as their catalytic cofactor. We previously described such a neofunctionalisation in Staphylococcus aureus, which altered a superoxide dismutase (SOD) enzyme from being an ancestral manganese-dependent (MnSOD) into an extant isozyme that can equally utilise either manganese or iron, termed cambialism (camSOD). Yet its unclear whether camSOD emergence involved selection solely for cofactor flexibility or whether other biochemical properties also diverged during neofunctionalisation. Here, we have investigated an independent biochemical property of the S. aureus SODs, their structural stability. We demonstrate that the neofunctionalised camSOD exhibits increased stability relative to the ancestral MnSOD. S. aureus camSOD is more resistant to both chemical and thermal unfolding in vitro. Crucially, while both isozymes possess a stable core at the heart of their fold, consisting of regions of the protein localised around the metal cofactor that resist hydrogen-deuterium exchange when exposed to isotopically labelled solvent, this core is larger and more exchange-resistant in camSOD than MnSOD. Thus, during the recent divergence of this SOD pair, two distinct biochemical properties have undergone substantial and rapid evolutionary change. This study paves the way for investigations of the structural and functional relationship between these properties, a SODs metal-preference and stability, and of how these properties were concomitantly selected during neofunctionalisation in the S. aureus lineage.

biochemistry↗

bioRxiv2025

A metalloenzyme's metal preference evolves through redox modulation driven by the cofactor's secondary coordination sphere

Changes in protein properties and functions are central to the evolution of life. Metalloproteins can evolve by changing their preference from one metal cofactor to another. Recently, we demonstrated that the widely distributed iron or manganese dependent superoxide dismutase (SodFM) family have undergone numerous metal-preference changes, including during evolutionary adaptation of pathogenic bacteria to altered metal availability within the host. Yet the underlying properties of metal-binding sites that control metalloenzyme metal-preference are unclear, and thus we lack an understanding of how enzymatic metal-preference can be re-shaped by evolution. Here, we used spectral features of bound iron or manganese, whose intensities reflect their oxidation state, to assess how their redox properties are manipulated during SodFM evolution. We systematically analysed the metal oxidation state across diverse SodFMs from multiple phylogenetic groups with different catalytic metal-preferences, including those known to have undergone evolutionary metal-preference switching. We observed a striking relationship between resting oxidation state and catalytic metal-preferences. Mutagenesis of second-sphere residues previously identified as determining metal preference revealed that they modulate metal-dependent activity and cofactor oxidation state in tandem, demonstrating these properties are linked. Together, these data argue that the differing SodFM metal preferences observed across the tree of life evolved through tuning of their redox properties by the secondary coordination sphere. This study gives insight into the process by which a metalloenzyme originally optimised for one metal cofactor can evolve a new metal preference, under suitable selection pressure, through re-optimisation of its active site for catalytic reactivity of the new metal cofactor. Significance statementMetal cofactors are needed by almost half of all enzymes. Catalytic metal-preference of metalloenzymes can evolve, for example to adapt to altered environmental metal availability. Yet, it is unclear how this evolutionary process occurs, enabling an active site optimised for one metal to change to become optimised for the new metal. Here, we have investigated this evolutionary process in a family of superoxide dismutase (SodFM) enzymes. We found that spectral features, which reflect the enzymes redox properties, of a diverse array of SodFMs with differing metal-preferences, and of mutated forms with artificially altered preferences, correlate with the metal-dependence of their activity. The data show that metal-preference change in SodFMs involves evolutionary tuning of the redox properties of SodFMs.

evolutionary biology↗

bioRxiv2025

A Zur-dependent regulatory RNA involved in maintaining zinc homeostasis in Staphylococcus aureus

Small regulatory RNAs (sRNAs) are key drivers of bacterial adaptation to environmental fluctuations, including iron and manganese restriction imposed by the host. This study explored the repertoire of sRNAs produced by the human pathogen Staphylococus aureus in response to metal limitation. Two sRNAs, S1077 and ZinS (RsaX20), regulated by zinc (Zn) availability, were identified. Further investigations revealed that, similar to the cnt operon from which it derives, S1077 synthesis is controlled by the transcription factors Zur and Fur. In contrast, zinS transcription is solely repressed by Zur. Amongst the ZinS targets are several Zn-dependent enzymes, such as the alcohol dehydrogenase Adh, whose synthesis is negatively regulated by ZinS. Loss of ZinS does not alter staphylococcal metal accumulation, suggesting a role in a Zn-sparing response. Remarkably, zinS also encodes a small peptide, ZinP. Genomic analysis suggests that the regulatory portion of ZinS emerged from the 3 untranslated region of zinP in S. aureus and closely related species after horizontal gene transfer from phylogenetically distant organisms. All our findings demonstrate that sRNAs also facilitate bacterial adaptation to Zn limitation, and that genetic exchange and subsequent neofunctionalization have enabled S. aureus to adapt to metal-restricted environments.

microbiology↗

bioRxiv2025

The gonococcal vaccine candidate antigen NGO1701 is a N. gonorrhoeae periplasmic copper storage protein

The increasing worldwide trend of antibiotic-resistant Neisseria gonorrhoeae strains highlights the urgent need for new therapeutic strategies against this sexually transmitted pathogen, including a gonococcal vaccine. We previously designed a bioinformatics-based candidate selection pipeline (CASS) and identified potential novel gonococcal vaccine targets among hypothetical proteins expressed during natural human infection. One of these candidates, NGO1701, is a predicted periplasmic four-helix bundle protein with amino acid sequence homology to the copper storage protein 1 (Csp1) from Methylosinus trichosporium OB3b. In this study, we confirmed that purified NGO1701 binds 15 Cu(I) ions per monomer in vitro, supporting its function as Csp in N. gonorrhoeae. Using a ngo1701 deletion mutant generated in N. gonorrhoeae F62, we investigated its role in bacteria physiology. We showed that ablation of Csp was not limiting for bacterial growth and fitness in vitro, but the {Delta}csp strain became significantly more susceptible to copper mediated toxicity. This phenotype was rescued by csp gene complementation, indicating a role in protection against copper toxicity. Our results indicate that Csp participates in periplasmic copper homeostasis in N. gonorrhoeae, buffering excess copper to reduce toxicity and playing a putative role in copper delivery to important copper-enzymes. Csp does not appear to be involved in bacterial host cell interaction and activation in vitro, since no difference in the ability of N. gonorrhoeae to adhere/invade epithelial cells or induce IL-8 secretion was reported among wild type, csp deletion mutant and complemented strains. Furthermore, sera from mice immunized with NGO1701 failed to recognize {Delta}csp by dot blot and ELISA, and the seras ability to kill N. gonorrhoeae was abrogated against {Delta}csp. However, both functions were restored after gene complementation, supporting the relevance of Csp as a potential vaccine target. Allelic analysis of Neisseria species revealed that this gene is absent in N. meningitidis, thus making it a gonococcal-specific target. Author SummaryCopper is essential for bacterial metabolism but can be toxic in excess. Here, we identify NGO1701 as a copper storage protein (Csp) in Neisseria gonorrhoeae, capable of sequestering Cu(I) ions. Deletion of csp led to increased copper sensitivity, while overexpression restored resistance, suggesting a role in copper homeostasis. The {Delta}csp mutant also showed reduced growth in cobalt and manganese, likely due to metal interference by copper toxicity. Beyond detoxification, Csp may supply copper to essential cuproenzymes like cytochrome cbb3 oxidase and nitric oxide reductase, which support bacterial survival under host-imposed stress. Although Csp is not required for N. gonorrhoeae host cell interactions, it is a strong immune target. Immune recognition of N. gonorrhoeae {Delta}csp by anti- NGO1701 mouse sera was nearly abolished and the serum bactericidal activity was abrogated compared to N. gonorrhoeae F62 wild type bacteria, highlighting Csps potential as a target for therapeutic or vaccine strategies against N. gonorrhoeae.

immunology↗