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Bacterial receptors feed into multiple signal transduction pathways that regulate a variety of cellular processes including gene expression, second messenger levels, and motility. Receptors are typically activated by signal binding to ligand-binding domains (LBDs). Cache domains are omnipresent LBDs found in bacteria, archaea, and eukaryotes, including humans. They form the predominant family of extracytosolic bacterial LBDs and were identified in all major receptor types. Cache domains are composed of either a single (sCache) or a double (dCache) structural module. The functional relevance of bimodular LBDs remains poorly understood. Here, we identify the PacF chemoreceptor in the phytopathogen Pectobacterium atrosepticum that recognizes formate at the membrane-distal module of its dCache domain, triggering chemoattraction. We further demonstrate that a family of formate-specific sCache domains has evolved from a dCache domain, exemplified by PacF, by losing the membrane-proximal module. By solving high-resolution structures of two family members in complex with formate, we show that the molecular basis for formate binding at sCache and dCache domains is highly similar, despite their low sequence identity. The apparent loss of the membrane-proximal module may be related to the observation that dCache domains bind ligands typically at the membrane-distal module, whereas studies have failed to find ligands bound in the membrane-proximal module. This work advances our understanding of signal sensing in bacterial receptors and suggests that evolution by reducing complexity may be a route for shaping diversity., Competing Interests: Competing interests statement:The authors declare no competing interest.

We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process. Of particular interest is the behavior of a double phenylalanine gate, which opens and closes according to the relative situations of haem and NADH within the protein pocket. This behavior suggests that the double phe-gate fulfills a regulatory role within the pocket, controlling the access of NADH to haem. Additionally, stopped-flow spectroscopy results provide kinetic comparisons between the wild-type and the selected mutants. We also present a fully resolved crystal structure for the F104AF199A HemS monomer, including its extensive loop, the first such structure to be completely resolved for HemS or any of its close homologues. The energy landscapes approach provided key information regarding the gating strategy employed by HemS, compensating for current limitations with conventional biophysical and molecular dynamics approaches. We propose that ELT become more widely used in the field, particularly in the investigation of the dynamics and interactions of weak-binding ligands, and for gating features, within protein cavities., (© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Periodontal diseases afflict 20-50% of the global population and carry serious health and economic burdens. Chronic periodontitis is characterized by inflammation of the periodontal pocket caused by dysbiosis. This dysbiosis is coupled with an increase in the population of Treponema denticola, a spirochete bacterium with high mobility and invasivity mediated by a number of virulence factors. One such virulence factor is TDE0362, a multidomain protein with a carboxy-terminal papain-superfamily cysteine protease (C0362). Most papain-superfamily cysteine proteases are produced as proenzymes with a prodomain that interacts with the prosegment binding loop (PBL), requiring proteolytic processing for full activation. Previous studies have indicated that C0362 is not produced as a proenzyme, suggesting an alternative regulatory mechanism. We previously determined the crystal structure of C0362 captured in an inactive conformation with an oxidized catalytic cysteine and a disordered PBL. In this follow-up study, we evaluated the active-site architecture and the PBL in two mutant (Y559A and C412S) structures and an inhibitor-bound (E64) structure to provide insight into the role that the PBL plays in the generation of active enzyme. Our results implicate Tyr559 as playing a critical role in the transition of the enzyme to an active state. We subsequently utilized the structural information to generate models of C0362 bound to human complement factors C3 and C4. Collectively, our results provide insight into the regulatory mechanism and putative substrate-binding interfaces of C0362, highlighting avenues of further research towards inhibition of this essential virulence factor.

Transition metals (e.g., Fe 2/3+ , Zn 2+ , Mn 2+ ) are essential enzymatic cofactors in all organisms. Their environmental scarcity led to the evolution of high-affinity uptake systems. Our research focuses on two bacterial manganese ABC importers, Streptococcus pneumoniae PsaBC and Bacillus anthracis MntBC, both critical for virulence. Both importers share a similar homodimeric structure, where each protomer comprises a transmembrane domain (TMD) linked to a cytoplasmic nucleotide-binding domain (NBD). Due to their size and slow turnover rates, the utility of conventional molecular simulation approaches to reveal functional dynamics is limited. Thus, we employed a novel, computationally efficient method integrating Gaussian Network Models (GNM) with information theory Transfer Entropy (TE) calculations. Our calculations are in remarkable agreement with previous functional studies. Furthermore, based on the calculations, we generated 10 point-mutations and experimentally tested their effects, finding excellent concordance between computational predictions and experimental results. We identified "allosteric hotspots" in both transporters, in the transmembrane translocation pathway, at the coupling helices linking the TMDs and NBDs, and in the ATP binding sites. In both PsaBC and MntBC, we observed bi-directional information flow between the two TMDs, with minimal allosteric transmission to the NBDs. Conversely, the NBDs exhibited almost no NBD-NBD allosteric crosstalk but showed pronounced information flow from the NBD of one protomer towards the TMD of the other protomer. This unique allosteric "footprint" distinguishes ABC importers of transition metals from other members of the ABC transporter superfamily establishing them as a distinct functional class. This study offers the first comprehensive insight into the conformational dynamics of these vital virulence determinants, providing potential avenues for developing urgently needed novel antibacterial agents., (© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Prokaryotic heme biosynthesis in Gram-positive bacteria follows the coproporphyrin-dependent heme biosynthesis pathway. The last step in this pathway is catalyzed by the enzyme coproheme decarboxylase, which oxidatively transforms two propionate groups into vinyl groups yielding heme b. The catalytic reaction cycle of coproheme decarboxylases exhibits four different states: the apo-form, the substrate (coproheme)-bound form, a transient three-propionate intermediate form (monovinyl, monopropionate deuteroheme; MMD), and the product (heme b)-bound form. In this study, we used cryogenic electron microscopy single-particle reconstruction (cryo-EM SPR) to characterize structurally the apo and heme b-bound forms of actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae. The flexible loop that connects the N-terminal and the C-terminal ferredoxin domains of coproheme decarboxylases plays an important role in interactions between the enzyme and porphyrin molecule. To understand the role of this flexible loop, we performed molecular dynamics simulations on the apo and heme b coproheme decarboxylase from Corynebacterium diphtheriae. Our results are discussed in the context of the published structural information on coproheme-bound and MMD-bound coproheme decarboxylase and with respect to the reaction mechanism. Having structural information of all four enzymatically relevant states helps in understanding structural restraints with a functional impact., (© 2025 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, specifically in the case of Gram-negative species. However, an increasing number of these pathogens employ a defense strategy that involves a relay of enzymes encoded by the pmrE (ugd) loci and the arnBCDTEF operon. The pathway modifies the lipid-A component of the outer membrane (OM) lipopolysaccharide (LPS) by adding a 4-amino-4-deoxy-l-arabinose (L-Ara4N) headgroup, which renders polymyxins ineffective. Here, we report the cryo-EM SPR structures of glycosyltransferase ArnC from Salmonella typhimurium determined in apo and UDP-bound forms at resolutions 2.75 Å and 3.8 Å, respectively. The structure of the ArnC protomer comprises three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a tetramer with C2 symmetry, where the C-terminal strand inserts into the adjacent protomer. This tetrameric state is further stabilized by two distinct interfaces formed by ArnC that form a network of hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilize the loop between residues H201 to S213, and part of the putative catalytic pocket formed by IH1 and IH2. The surface property analysis revealed a hydrophobic cavity formed by TM1 and TM2 in the apo state, which is disrupted upon UDP binding. The comparison of ArnC structures to their homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity., (© 2025 The Protein Society.)

The microbial manganese removal process is believed to consist of the catalytic oxidation of Mn(II) by manganese oxidase. In this study, the multicopper oxidase CopA was purified and exhibited high manganese oxidation activity in vitro, and it was found that Cu(II) can significantly enhance its manganese oxidation activity. Gene site-directed mutagenesis was used to mutate four conserved copper binding sites of CopA to obtain four mutant strains. The manganese removal efficiencies of the four strains were determined, and it was found that H120 is the catalytically active site of CopA. The loss of Cu(II) and the mutation of the conserved copper binding site H120 resulted in the loss of ethoxyformyl and quinone modifications, a reduction in the number of modifications, and a change in the position of modifications, eventually causing a decrease in protein activity from 85.87% to 70.1%. These results reveal that Cu(II) and H120 play an indispensable role in manganese oxidation by the multicopper oxidase CopA. X-ray photoelectron spectroscopy (XPS) analysis indicates that biogenic manganese oxides produced by strains and by CopA were both composed of MnO 2 and Mn 3 O 4 and that the average valence of Mn was 3.2., (© 2024 Wiley Periodicals LLC.)

Tuberculosis is one of the most ancient infectious diseases known to mankind predating upper Paleolithic era. In the current scenario, treatment of drug resistance tuberculosis is the major challenge as the treatment options are limited, less efficient and more toxic. In our study we have developed an atom based 3D QSAR model, statistically validated sound with R2 > 0.90 and Q2 > 0.72 using reported direct inhibitors of InhA (2018-2022), validated by enzyme inhibition assay. The model was used to screen a library of 3958 molecules taken from Binding DB and candidates molecules with promising predicted activity value (pIC50) > 5) were selected for further analyzed screening by using molecular docking, ADME profiling and molecular dynamic simulations. The lead molecule, ZINC11536150 exhibited good docking score (glideXP = -11.634 kcal/mol) compared to standard triclosan (glideXP =  -7.129 kcal/mol kcal/mol) and through molecular dynamics study it was observed that the 2nv6-complex of ZINC11536150 with Mycobacterium tuberculosis InhA (PDB entry: 2NV6) complex remained stable throughout the entire simulation time of 100 ns.Communicated by Ramaswamy H. Sarma.

The E. coli FtsZ dimer was studied to gain insights into FtsZ protofilament formation. In the simulation study of the M. janaschii dimer it was found that the monomer-monomer contacts in the GDP bound dimer is lower which results in the high curvature of the GDP bound protofilaments. In this study, the E. coli FtsZ dimer was simulated. The initial structure was obtained from our previous study in which we had simulated the E. coli FtsZ monomer with its C-terminal IDR (Intrinsically Disordered Region). The M. janaschii FtsZ dimer subunit contacts were used as the starting configuration. Simulations of the dimer were performed with GTP and with GDP. It is found that the central helix H5 closes by about 15 degrees in the simulation with GTP than in the simulation with GDP. The C-terminal IDR and the C-terminal domain region between SC2 and HC2 are found to have much high flexibility and hence exhibit domain motion.Communicated by Ramaswamy H. Sarma.

Protein-protein interactions (PPIs) have been recognized as a promising target for the development of new drugs, as proved by the growing number of PPI modulators reaching clinical trials. In this context, peptides represent a valid alternative to small molecules, owing to their unique ability to mimic the target protein structure and interact with wider surface areas. Among the possible fields of interest, bacterial PPIs represent an attractive target to face the urgent necessity to fight antibiotic resistance. Growing attention has been paid to the YgjD/YeaZ/YjeE complex responsible for the essential t 6 A 37 tRNA modification in bacteria. We previously identified an α-helix on the surface of Pseudomonas aeruginosa YeaZ, crucial for the YeaZ-YeaZ homodimer formation and the conserved YeaZ-YgjD interactions. Herein, we present our studies for impairing the PPIs involved in the formation of the YeaZ dimers through synthetic peptide derivatives of this helical moiety, both in vitro with purified components and on P. aeruginosa cells. Our results proved the possibility of targeting those PPIs which are usually essential for protein functioning and thus are refractory to mutational changes and antibiotic resistance development., (© 2024 The Author(s). Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.)

The plasmid-encoded mobile colistin resistance enzyme (MCR) is challenging the clinical efficacy of colistin as a last-resort antibiotic against multidrug-resistant bacteria. This transferase catalyzes the addition of positively charged phosphoethanolamine to lipid A, and its catalytic domain in the periplasm has been elucidated. To date, there are many works on the catalytic domain and function of this enzyme class. However, the roles of unreported soluble or inter-membrane domains remain undefined, which might cause an inaccurate or even incorrect understanding of substrate recognition and binding. In this review, MCR-1 is first compared and analyzed from the perspective of the full-length alpha-fold MCR-1. Specifically, some disputed issues, especially in its architecture and catalytic mechanism are discussed independently. Meanwhile, the structure-based insights into MCRs variants, their evolutions, and the balance between colistin-resistance and survival costs, are also critically analyzed. Importantly, by comparing it with the full-length MCR-1, several potential pockets for drug design have been re-identified. Finally, recent advancements in inhibitors targeting MCR-1 are also in-depth summarized. These details offer a new perspective on MCRs and serve as a valuable foundation for drug development., Competing Interests: Declaration of Competing Interest The author declares no competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Acetolactate synthase (ALS) is an essential enzyme involved in the biosynthesis of platform chemicals acetoin and 2,3-butanediol in several microorganisms. In this study, we investigated the catalytic differences among three bacterial ALSs involved in the ligation of two molecules of pyruvate or 2-ketobutyrate. Based on the findings, we predicted three amino acid residues in each enzyme that caused a discrepancy in accordance with the multi-sequence alignment and molecular docking experiments: I398, A402, and T480 in Bacillus subtilis ALS; V400, Y404, and S482 in Listeria seleigeri serovar 1/2b ALS; and M394, H398, and G476 in Klebsiella pneumoniae ALS. Subsequently, we mutually mutated the residues in the three ALSs. The data obtained confirmed our inference that these three residues in each enzyme are truly correlated with substrate recognition, particularly in recognizing compounds that are larger than pyruvate, such as 2-ketobutyrate, benzaldehyde, and nitrosobenzene. This study further clarifies the biochemical traits of ALSs derived from various bacteria and expands the scope of ALS research., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Metal ion coordination is crucial in bacterial metabolism, while divalent metal ions serve as essential cofactors for various enzymes involved in cellular processes. Therefore, bacteria have developed sophisticated regulatory mechanisms to maintain metal homeostasis. These involve protein interactions for metal ion uptake, efflux, intracellular transport, and storage. Staphylococcus aureus, a member of the commensal flora, colonizes the anterior nares and skin harmlessly but can cause severe illness. MntH transporter is responsible for acquiring divalent metal ions necessary for metabolic functions and virulence. It is a 450-amino-acid protein analogous to Nramp1 (Natural Resistance-Associated Macrophage Protein 1) in mammals. Herein, the coordination modes of copper(II), iron(II), and zinc(II) ions with select fragments of the MntH were established employing potentiometry, mass spectrometry, and spectroscopic methods. Four model peptides, MNNKRHSTNE-NH 2 , Ac-KFDHRSS-NH 2 , Ac-IMPHNLYLHSSI-NH 2 , and Ac-YSRHNNEE-NH 2 , were chosen for their metal-binding capabilities and examined to determine their coordination properties and preferences. Our findings suggest that under physiological pH conditions, the N-terminal fragment of MntH demonstrates the highest thermodynamic stability with copper(II) and iron(II) ions. Furthermore, a comparison with other peptides from the S. aureus FeoB transporter indicates different binding affinities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The acquisition of ferrous iron (Fe 2+ ) is crucial for the survival of many pathogenic bacteria living within acidic and/or anoxic conditions such as Vibrio cholerae, the causative agent of the disease cholera. Bacterial pathogens utilize iron as a cofactor to drive essential metabolic processes, and the primary prokaryotic Fe 2+ acquisition mechanism is the ferrous iron transport (Feo) system. In V. cholerae, the Feo system comprises two cytosolic proteins (FeoA, FeoC) and a complex, polytopic transmembrane protein (FeoB) that is regulated by an N-terminal soluble domain (NFeoB) with promiscuous NTPase activity. While the soluble components of the Feo system have been frequently studied, very few reports exist on the intact membrane protein FeoB. Moreover, FeoB has been characterize almost exclusively in detergent micelles that can cause protein misfolding, disrupt protein oligomerization, and even dramatically alter protein function. As many of these characteristics of FeoB remain unclear, there is a critical need to characterize FeoB in a more native-like lipid environment. To address this unmet need, we employ styrene-maleic acid (SMA) copolymers to isolate and to characterize V. cholerae FeoB (VcFeoB) encapsulated by a styrene-maleic acid lipid particle (SMALP). In this work, we describe the development of a workflow for the expression and the purification of VcFeoB in a SMALP. Leveraging mass photometry, we explore the oligomerization of FeoB in a lipid bilayer and show that the VcFeoB-SMALP is mostly monomeric, consistent with our previous oligomerization observations in surfo. Finally, we characterize the NTPase activity of VcFeoB in the SMALP and in a detergent (DDM), revealing higher NTPase activity in the presence of the lipid bilayer. When taken together, this report represents the first characterization of any FeoB in a native-like lipid bilayer and provides a viable approach for the future structural characterization of FeoB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As toxic pollutants, n -alkanes are pervasively distributed in most environmental matrices. Although the alkane monooxygenase AlmA plays a critical role in the metabolic pathway of solid long-chain n -alkanes (≥C 20 ) that are extremely difficult to degrade, the mechanism regulating this process remains unclear. Here, we characterized the function of AlmA in Acinetobacter venetianus RAG-1, which was mainly involved in the degradation of long-chain n -alkanes (C 26 -C 38 ), among which, n -C 32 induced the almA promoter activity most. APR1 ( A lmA P ositive R egulator) that it is an AraC/XylS-type transcription regulator, a potential transcriptional regulator of almA , was screened by DNA-pull down, which was determined by conserved domain analysis. The deletion of apR1 severely reduced the capacity of strain RAG-1 to utilize long-chain n -alkanes (C 22 -C 38 ), indicating the involvement of APR1 in n -alkanes degradation. The results of the APR1-dependent reporter system, electrophoretic mobility shift assay, and microscale thermophoresis further proved that APR1 was able to directly bind to the almA promoter region, thus activating the almA transcription. Furthermore, APR1 could inhibit self-expression through autoregulation in the absence of long-chain n -alkanes. n -C 32 acted as a ligand of APR1, and the amino acid residues Val10, Gln50, Ala99, and Ile106 at the N-terminus of APR1 were necessary for binding n -C 32 . In addition, the key amino acid residues of APR1 within the C-terminal helix-turn-helix motif that bound to the downstream promoter region were confirmed by multiple sequence alignment and site-directed mutagenesis. The homologs of AlmA and APR1 shared a similar evolutionary course in the Proteobacteria ; thus, this mode of regulation might be relatively conserved., Importance: The extreme hydrophobicity of long-chain n -alkanes ({greater than or equal to}C 20 ) presents a significant challenge to their degradation in natural environments. It is, therefore, imperative to elucidate the regulatory mechanisms of the metabolic pathways of long-chain n -alkanes, which will be of great significance for the future application of hydrocarbon-degrading bacteria to treat oil spills. However, the majority of current studies have focused on the regulatory mechanisms of short- and medium-chain n -alkanes, with long-chain n -alkanes receiving comparatively little attention. In this study, we identified APR1, a transcriptional regulator of the alkane monooxygenase AlmA in Acinetobacter venetianus RAG-1, and characterized its function and regulatory mechanism. In the presence of ligand n -C 32 , APR1 could directly activate the transcription of almA , which was involved in the n -C 32 metabolism. The amino acid residue unique to the C-terminal DNA-binding domain of AraC/XylS type n -alkanes transcription regulators was also identified. These findings further improved our understanding of the long-chain n -alkanes degradation mechanism, which is important for the management of petroleum pollution., Competing Interests: The authors declare no conflict of interest.

MutT proteins are Nudix hydrolases characterized by the presence of a Nudix box, GX5EX7REUXEEXGU, where U is a bulky hydrophobic residue and X is any residue. Major MutT proteins hydrolyze 8-oxo-(d)GTP (8-oxo-GTP or 8-oxo-dGTP) to the corresponding 8-oxo-(d)GMP, preventing their incorporation into nucleic acids. Mycobacterial MutT1 comprises an N-terminal domain (NTD) harboring the Nudix box motif, and a C-terminal domain (CTD) harboring the RHG histidine phosphatase motif. Interestingly, unlike other MutTs, the MutT1 hydrolyses the mutagenic 8-oxo-(d)GTP to the corresponding 8-oxo-(d)GDP. Nucleoside diphosphate kinase (NDK), a conserved protein, carries out reversible conversion of (d)NDPs to (d)NTPs through phospho-NDK (NDK- Pi ) intermediate. Recently, we showed that NDK- Pi converts 8-oxo-dGDP to 8-oxo-dGTP and escalates A to C mutations in a MutT-deficient Escherichia coli . We now show that both Mycobacterium tuberculosis MutT1 and Mycobacterium smegmatis MutT1, through their NTD (Nudix hydrolase motifs) function as protein phosphatase to regulate the levels of NDK- Pi and prevent it from catalyzing conversion of (d)NDPs to (d)NTPs (including conversion of 8-oxo-dGDP to 8-oxo-dGTP). To corroborate this function, we show that Msm MutT1 decreases A to C mutations in E. coli under the conditions of Eco NDK overexpression.IMPORTANCEMutT proteins, having a Nudix box domain, hydrolyze the mutagenic 8-oxo-dGTP to 8-oxo-dGMP. However, mycobacterial MutT (MutT1) comprises an N-terminal domain (NTD) harboring a Nudix box, and a C-terminal domain (CTD) harboring an RHG histidine phosphatase. Unlike other MutTs, mycobacterial MutT1 hydrolyses 8-oxo-dGTP to 8-oxo-dGDP. Nucleoside diphosphate kinase (NDK), a conserved protein, converts 8-oxo-dGDP to 8-oxo-dGTP through phospho-NDK (NDK- Pi ) intermediate and escalates A to C mutations. Here, we show that the mycobacterial MutT1 is unprecedented in that its NTD (Nudix box), functions as protein phosphatase to regulate NDK- Pi levels and prevents it from converting dNDPs to dNTPs (including 8-oxo-dGDP to 8-oxo-dGTP conversion). In addition, mycobacterial MutT1 decreases A to C mutations in Escherichia coli under the conditions of NDK overexpression., Competing Interests: The authors declare no conflict of interest.

Background: Bacillus inaquosorum strains is widely recognized for their plant-growth-promoting and biocontrol capabilities, yet their roles in protease production remain unclear. The present study aimed to comprehensively assess the protease-producing performance of B. inaquosorum strain E1-8, at the same time as exploring the novel application of agricultural Bacillus proteases in the preparation of protein hydrolysates for fresh-cut fruits preservation., Results: First, genomic sequencing revealed the diversity of E1-8 proteases, indicating 15 putative extracellular proteases. Subsequently, the fermentation conditions for E1-8 protease production were optimized, with sweet potato powder and soybean meal identified as the most suitable carbon and nitrogen sources, respectively, resulting in a maximum protease activity of 321.48 U mL -1 . Upon culturing the strain under these optimized conditions, only an S8 family serine protease and an M48 family metalloprotease were revealed by secretomic analysis and protease inhibitor assays. Additionally, the optimal protease conditions for generating protein hydrolysates from soy, pea, fish and porcine proteins were determined. The molecular weight of the hydrolysates primarily ranged from 2000 to 180 Da, with a total of 17 amino acids identified. The application of these hydrolysates demonstrated a 2,2-diphenyl-1-picrylhydrazyl (i.e. DPPH) scavenging activity ranging from 58.64% to 84.12%, significantly reducing of the melting peaks and the freezing points. Furthermore, the browning index of apple slices stored at 4 °C decreased by 14.81% to 22.15% on the second day, and similar effects were observed in fresh-cut banana stored at 4 °C for 7 days., Conclusion: The protein hydrolysates obtained exhibit remarkable antioxidant, antifreeze and anti-browning properties for fresh-cut fruits. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Dispersal plays a crucial role in the development and ecology of biofilms. While extensive studies focused on elucidating the molecular mechanisms governing this process, few have characterized the associated temporal changes in composition and structure. Here, we employed solid-state nuclear magnetic resonance (NMR) techniques to achieve time-resolved characterization of Bacillus subtilis biofilms over a 5-day period. The mature biofilm, established within 48 h, undergoes significant degradation in following 72 h. The steepest decline of proteins precedes that of exopolysaccharides, likely reflecting their distinct spatial distribution. Exopolysaccharide sugar units display clustered temporal patterns, suggesting the presence of distinct polysaccharide types. A sharp rise in aliphatic carbon signals on day 4 probably corresponds to a surge in biosurfactant production. Different dynamic regimes respond differently to dispersal: the mobile domain exhibits increased rigidity, while the rigid domain remains stable. These findings provide novel insights and perspectives on the complex process of biofilm dispersal., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Lipoteichoic acid synthase (LtaS) is crucial for the biosynthesis of lipoteichoic acid (LTA) in lactic acid bacteria (LAB), where LTA plays a key role in bacterial adhesion, immune modulation, and maintaining cell integrity. This study explores the regulation of LtaS activity in Lactiplantibacillus plantarum , examining the effects of factors such as temperature, pH, and metal ions on enzyme activity. Molecular docking was used to identify critical amino acids at the enzyme's active site, and site-directed mutagenesis confirmed the role of five key residues (Glu-259, Thr-303, Asn-353, Arg-360, and His-420) in LtaS activity. Among them, Thr-303 plays a pivotal role, followed by Glu-259 and His-420. Conservation analysis revealed that these active-site residues are highly conserved across LAB species. These findings provide valuable insights into the functional properties of LtaS, offering potential for enhancing the efficacy of LAB-based probiotics and improving their therapeutic benefits in health applications.

Phycobilisomes (PBS) are the major photosynthetic light-harvesting complexes in cyanobacteria and red algae. While the structures of PBS have been determined in atomic resolutions, how PBS are attached to the reaction centers of photosystems remains less clear. Here, we report that a linker protein (LcpA) is required for the attachment of PBS to photosystem II (PSII) in the cyanobacterium Synechococcus sp. PCC 7002. We also report that the PB-loop of PBS, which is located within the α-APC domain of ApcE, is required for the attachment of PBS to PSII. Deletion of either PB-loop or the gene A0913 led to a decreased rate of photoautotrophic growth under illumination of green light, which is preferentially absorbed by PBS. A double mutant lacking the PB-loop and A0913 (ΔPBL-0913) showed a complete inhibition of O 2 evolution under the 590 nm light and could not grow under green light illumination. While assembled PBS could be isolated from ΔPBL-0913, the energy transfer from its PBS to PSII was blocked as measured by fluorescence induction. Photobleaching with intact cells showed that the PBS movement speed in ΔPBL-0913 was 2.5 times as fast as that of the wild type, suggesting that association of its PBS with thylakoids was weakened significantly. The pull-down and coimmunoprecipitation results showed that the LcpA interacts with the CP47 subunit of PSII through its N-terminal region and interacts with ApcB of PBS through its C-terminal α-helix motif. Our results provide insights into the molecular mechanism of PBS-PSII association and shed light on excitation energy transfer from PBS to PSII., Competing Interests: Competing interests statement:The authors declare no competing interest.

Malachite green (MG) is used as a dye for materials such as wood, cotton, and nylon, and is used in aquaculture to prevent fungal and protozoan diseases. However, it is highly toxic, with carcinogenic, mutagenic, and teratogenic properties, resulting in bans worldwide. Despite this, MG is still frequently used in many countries due to its efficacy and economy. MG is persistent in the environment and so requires degradative intervention. In this work we isolated Bacillus pacificus ROC1 strain from a salt flat in Pakistan that had the ability to aerobically detoxify MG, as determined by bacterio- and phyto-toxicity assays. We demonstrate immobilized B. pacificus ROC1 can effectively detoxify MG, which highlights a potential method for its biodegradation. Genomic sequencing identified three candidate azo-reductases within B. pacificus ROC1 that could be responsible for the MG-degrading activity. These were cloned, expressed and purified from Escherichia coli, with one (AzrC), catalyzing the reduction of MG to leuco-MG in vitro. AzrC was crystallised and MG was captured within the active site in a Michaelis complex, providing structural insight into the reduction mechanism. Altogether, this work identifies a bacterium capable of aerobically degrading a major industrial pollutant and characterizes the molecular basis for this activity., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Cell movement on surfaces relies on focal adhesion complexes (FAs), which connect cytoskeletal motors to the extracellular matrix to produce traction forces. The soil bacterium Myxococcus xanthus uses a bacterial FA (bFA), for surface movement and predation. The bFA system, known as Agl-Glt, is a complex network of at least 17 proteins spanning the cell envelope. Despite understanding the system dynamics, its molecular structure and protein interactions remain unclear. In this study, we utilize AlphaFold to generate models based on the known interactions and dynamics of gliding motility proteins. This approach provides us with a comprehensive view of the interactions across the entire complex. Our structural insights show the connection of essential functional modules throughout the cell envelope and offer an inspiring view of the force transduction mechanism from the inner molecular motor to the exterior of the cell., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The sulfosugar sulfoquinovose (SQ) is catabolized through the sulfoglycolytic Entner-Doudoroff pathway, beginning with the oxidation of SQ to sulfogluconolactone by SQ dehydrogenase. We present a comprehensive structural and kinetic characterization of Pseudomonas putida SQ dehydrogenase (PpSQDH). PpSQDH is a tetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) superfamily with a strong preference for NAD+ over NADP+. Kinetic analysis revealed a rapid equilibrium ordered mechanism in which the NAD+ cofactor is the first substrate to bind, and NADH is the last product to dissociate. Structural studies revealed a homotetrameric structure in solution and crystals, involving cross-subunit interactions in which the C-terminus residue (Gln260) inserts into the diagonally opposite subunit to form part of the second shell of residues lining the active site. Complexes of PpSQDH with SQ or NAD+ provide insight into the recognition of SQ and together with the kinetic analysis allow the proposal of a catalytic reaction mechanism. Our findings illuminate the mechanism of SQ degradation and the evolution of the SDR superfamily for organosulfonate catabolism., (© 2025 The Author(s).)

We present a neo-Hookean elasticity theory for hybrid mechano-active hydrogels, integrating motor proteins into polymer meshes to create composite materials with active softening due to modulable chain overlaps. Focusing on polyacrylamide (PA) hydrogels embedded with FtsZ, a bacterial cytokinetic protein powered by GTP, we develop a multiscale model using microscopic Flory theory of rubbery meshes through mesoscopic De Gennes' scaling concepts for meshwork dynamics and phenomenological Landau's formalism for second-order phase transitions. Our theoretical multiscale model explains the active softening observed in hybrid FtsZ-PA hydrogels by incorporating modulable meshwork dynamics, such as overlapping functionality and reptation dynamics, into an active mean-field of unbinding interactions. The novel FtsZ-based metamaterial and companion multiscale theory offer insights for designing, predicting, and controlling complex active hydrogels, with potential applications in technology and biomedicine.

Streptomyces griseolus CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs. We successfully crystallized complexes of wild-type CYP105A1 (WT) and the R84A variant with diclofenac (DIF) or flufenamic acid (FLF). In the WT, the carboxyl group of DIF formed a charged hydrogen bond with Arg84. In contrast, in R84A, the carboxyl group formed two bidentate charged hydrogen bonds with Arg73. The C4' atom of the benzene ring of DIF, which undergoes hydroxylation by WT and R84A, was positioned approximately 4 Å from the heme iron. Binding of FLF was nearly the same in both WT and R84A. The carboxyl group of FLF formed charged hydrogen bonds with Arg73. In both WT and R84A, FLF appeared to be fixed by this charged hydrogen bonding with Arg73 during the reaction, and the C4' atom, which undergoes hydroxylation, must face the heme iron. Thus, the dihedral angles of the two N-C bonds connecting the two benzene rings of FLF needed to rotate by 78° and -71°, respectively. The temperature factors of the F-G loop, helix F, and helix G of R84A were remarkably higher than those of WT. This suggests that these regions in R84A are much more flexible compared to those of WT, which may consequently affect substrate binding and product release.

The accumulation-associated protein (Aap) is the primary determinant of Staphylococcus epidermidis device-related infections. The B-repeat superdomain is responsible for intercellular adhesion that leads to the development of biofilms occurring in such infections. It was recently demonstrated that Zn-induced B-repeat assembly leads to formation of functional amyloid fibrils, which offer strength and stability to the biofilm. Rigorous biophysical studies of Aap B-repeats from S. epidermidis strain RP62A revealed Zn-induced assembly into stable, reversible dimers and tetramers, prior to aggregation into amyloid fibrils. Genetic manipulation is not tractable for many S. epidermidis strains, including RP62A; instead, many genetic studies have used strain 1457. Therefore, to better connect findings from biophysical and structural studies of B-repeats to in vivo studies, the B-repeat superdomain from strain 1457 was examined. Differences between the B-repeats from strains RP62A and 1457 include the number of B-repeats, which has been shown to play a critical role in assembly into amyloid fibrils, as well as the distribution of consensus and variant B-repeat subtypes, which differ in assembly competency and thermal stability. Detailed investigation of the Zn-induced assembly of the full B-repeat superdomain from strain 1457 was conducted using analytical ultracentrifugation. Whereas the previous construct from RP62A (Brpt5.5) formed a stable tetramer prior to aggregation, Brpt6.5 from 1457 forms extremely large stable species on the order of ≈28-mers, prior to aggregation into similar amyloid fibrils. Our data suggest that both assembly pathways may proceed through the same mechanism of dimerization and tetramerization, and both conclude with the formation of amyloid-like fibrils. Discussion of assembly behavior of B-repeats from different strains and of different length is provided with considerations of biological implications., Competing Interests: Declaration of interests A.B.H. served as a Scientific Advisory Board member for Hoth Therapeutics, Inc., holds equity in Hoth Therapeutics and Chelexa BioSciences, LLC, and was a co-inventor on seven patents broadly related to the subject matter of this work., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Cyanobacteria are responsible for up to 80% of aquatic carbon dioxide fixation and have evolved a specialized carbon concentrating mechanism to increase photosynthetic yield. As such, cyanobacteria are attractive targets for synthetic biology and engineering approaches to address the demands of global energy security, food production, and climate change for an increasing world's population. The bicarbonate transporter BicA is a sodium-dependent, low-affinity, high-flux bicarbonate symporter expressed in the plasma membrane of cyanobacteria. Despite extensive biochemical characterization of BicA, including the resolution of the BicA crystal structure, the dynamic understanding of the bicarbonate transport mechanism remains elusive. To this end, we have collected over 1 ms of all-atom molecular dynamics simulation data of the BicA dimer to elucidate the structural rearrangements involved in the substrate transport process. We further characterized the energetics of the transition of BicA protomers and investigated potential mutations that are shown to decrease the free energy barrier of conformational transitions. In all, our study illuminates a detailed mechanistic understanding of the conformational dynamics of bicarbonate transporters and provides atomistic insights to engineering these transporters for enhanced photosynthetic production., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Mycobacterium tuberculosis (Mtb) is a leading cause of death, with an escalating global occurrence of drug-resistant infections that are partially attributed to cell wall mycolic acids derived from type II fatty acid biosynthesis (FAS-II). Here, the central acyl carrier protein, AcpM, contributes to the regulation of complex and specific protein-protein interactions (PPIs), though the orchestration of these events remain largely unresolved due to unique features of AcpM. Limitations include complexities in generating modified AcpM in a single state. Herein, we report a streamlined method to generate homogeneous samples of modified AcpM for applications in structure and functional studies. We apply these to generate solvatochromic labeled crypto -AcpM, where fluorescence response reports cargo sequestration and chain flipping upon interaction with four FAS-II enzymes. We find an increased fluorescence in a truncated form, AcpM80, indicating that the 35-residue C-terminus is involved in modulating the chemical environment surrounding the substrate and contributing to the regulation of PPIs. This study establishes an efficient chemo-enzymatic strategy to generate AcpM analogs for biophysical studies to aid in understanding the processes driving Mtb pathogenicity and drug resistance.

The Orange Carotenoid Protein (OCP) is a unique water-soluble photoactive protein that plays a critical role in regulating the balance between light harvesting and photoprotective responses in cyanobacteria. The challenge in understanding OCP´s photoactivation mechanism stems from the heterogeneity of the initial configurations of its embedded ketocarotenoid, which in the dark-adapted state can form up to two hydrogen bonds to critical amino acids in the protein's C-terminal domain, and the extremely low quantum yield of primary photoproduct formation. While a series of experiments involving point mutations within these contacts helped us to identify these challenges, they did not resolve them. To overcome this, we shifted from classical mutagenesis to the translational introduction of non-canonical amino acid residues into the OCP structure. In this work, we demonstrate that replacing a single meta-hydrogen in tyrosine-201 with a halogen atom (chlorine, bromine, or iodine) leads to targeted modifications in the keto-carotenoid-protein matrix interaction network, both in the dark-adapted state and upon photoactivation. We found that such atomic substitutions allow us to effectively weaken key hydrogen bonds without disrupting protein folding, thereby increasing the yield of OCP photoactivation products. Such genetically encoded chemical modification of individual atoms and their systematic in situ variation in complex protein structures establishes a foundation for transforming OCP into a practical tool for optogenetics and other applications., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature B.V.)

The flagellar MS-ring is the initial template for flagellar assembly and houses the flagellar protein export complex. The MS-ring has three parts of different symmetries within the ring structure by assembly of FliF subunits in two different conformations with distinct arrangements of three ring-building motifs, RBM1, RBM2, and RBM3. However, it remains unknown how these symmetries are generated. A combination of cryoEM structure and structure-based mutational analyses demonstrates that the well-conserved DQxGxxL motif in the RBM2-RBM3 hinge loop allows RBM2 to take two different orientations relative to RBM3. Of 34 FliF subunits of the MS-ring in the basal body, 23 RBM2 domains form an inner ring with a central pore that accommodates the flagellar protein export complex, and the remaining 11 RBM2 domains form 11 cog-like structures together with RBM1 domains just outside the inner RBM2-ring. We propose that a dimer of FliF with two different conformations initiates MS-ring assembly., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Streptococcus pneumoniae (SPN) is a significant pathogen causing pneumonia and meningitis, particularly in vulnerable populations like children and the elderly. Available pneumonia vaccines have limitations since they only cover particular serotypes and have high production costs. The emergence of antibiotic-resistant SPN strains further underscores the need for a new, cost-effective, broad-spectrum vaccine. Two potential vaccine candidates, CbpA and PspA, were identified, and their B-cell, CTL, and HTL epitopes were predicted and connected with suitable linkers, adjivant and PADRE sequence. The vaccine construct was found to be antigenic, non-toxic, non-allergenic, and soluble. The three-dimensional structure of the vaccine candidate was built and validated. Docking analysis of the vaccine candidate by ClusPro demonstrated robust and stable binding interactions between the MEV and toll-like receptor 4 in both humans and animals. The iMOD server and Amber v.22 tool has verified the stability of the docking complexes. GenScript server confirmed the high efficiency of cloning for the construct and in-silico cloning into the pET28a (+) vector using SnapGene, demonstrating successful translation of the epitope region. Immunological responses were shown to be enhanced by the C-IMMSIM server. This study introduced a strong peptide vaccine candidate that has the potential to contribute to the development of a rapid and cost-effective solution for combating SPN. However, experimental verification is necessary to evaluate the vaccine's effectiveness., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2025 Nahian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The probiotic Bifidobacterium breve is known for its efficient conjugated linoleic acid (CLA) conversion, yet their CLA conversion pathway remains underexplored. This study investigated B. breve JKL2022 for its CLA conversion in actively growing cells, washed cell states, and in crude protein extracts. Moreover, the study aimed to confirm the CLA-converting enzyme in strain JKL2022 and optimize its purification through heterologous expression of fusion proteins (LAI_sGFP and MBP_LAI). JKL2022 exhibited superior CLA conversion compared to genetically similar B. breve strains (JCM7017, JCM7019, JCM1192, and JCM1273), particularly the observed CLA conversion in washed cells (60.14 ± 7.60%) and crude protein fractions (96.11 ± 6.63%). The multipass transmembrane linoleic acid isomerase (LAI) was cloned into the E. coli BL21(DE3) as free LAI or modified with superfolder-GFP or MBP tags and expressed with 0.01 mM IPTG at 37 °C, resulting in highly active protein fractions. LAI was characterized by predictive modeling, molecular docking, and phylogenetic analyses. Moreover, reverse transcription-quantitative PCR analysis revealed upregulation (20-140× higher expression) of lai in JKL2022 compared with that in the JCM strains. Nevertheless, upscaling the production and purification of LAI for downstream applications remains a challenge, primarily because of their membrane-spanning configuration.

d-Tagatose, a rare sugar endowed with a low-calorie property, superior taste quality, and probiotic functionality, has garnered significant research attention. However, the prevailing biological production methods relying on β-galactosidase and l-arabinose isomerase face challenges including high cost and suboptimal conversion efficiency. Consequently, it is of great research significance to find efficient alternative routes for d-tagatose synthesis. Previously, Thermotoga petrophila tagaturonate 3-epimerase was modified to function as tagatose 4-epimerase (T4E) enabling the direct conversion of d-fructose to d-tagatose. In this study, T4E was further engineered through directed evolution, specifically targeting the enhancement of its thermostability for application. This endeavor yielded promising T4E variants with superiority over those of the original enzyme. T4E I430P exhibits a half-life ( t 1/2 ) at 70 °C that is 1.83-fold that of T4E, with an increased melting temperature ( T m ) of 5.1 °C compared to T4E. Additionally, T4E G90S/T272A/I430P demonstrated a 21.4% increase in specific activity compared to T4E. At 70 °C, its t 1/2 was 1.69-fold that of T4E, and its T m is 2.9 °C higher than T4E. Furthermore, whole-cell immobilization integrating these engineered T4E variants into a robust biocatalytic system was employed. This innovative approach not only underscores the practical feasibility of modifying enzymes through directed evolution but also establishes a foundation for the cost-effective, large-scale production of d-tagatose.

Many plant proteins are amphiphilic molecules that can adsorb to air-water interfaces and form protective coatings around gas bubbles. In this study, the composition, structure, physicochemical properties, air-water interfacial properties, and foaming properties of 16 plant and microbial proteins were characterized. We found a correlation between the composition, structure, physicochemical properties, and foaming properties of the proteins. The foaming capacity of them showed a highly significant positive correlation (p ≤ 0.01) with their foaming stability, α-helix content, surface hydrophobicity, and free sulfhydryl content. The foaming capacity and foaming stability showed highly significant negative correlations with disulfide bond content (p ≤ 0.01). We found wheat gluten protein (WGP) and mung bean protein (MBP) had higher foaming capacity (102.67 ± 8.08 % and 89.33 ± 4.72 %), which could be attributed to higher surface hydrophobicity (179.68 ± 1.40 and 130.28 ± 1.41) and larger contact angle (82.369 ± 0.016° and 82.949 ± 0.228°)., Competing Interests: Declaration of competing interest We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, “Interfacial and Foaming Properties of Plant and Microbial Proteins: Comparison of Structure-Function Behavior of Different Proteins”., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The loading of the bacterial replicative helicase DnaB is an essential step for genome replication and depends on the assistance of accessory proteins. Several of these proteins have been identified across the bacterial phyla. DciA is the most common loading protein in bacteria, yet the one whose mechanism is the least understood. We have previously shown that DciA from Vibrio cholerae is composed of a globular domain followed by an unfolded extension and demonstrated its strong affinity for DNA. Here, we characterize the condensates formed by VcDciA upon interaction with a short single-stranded DNA substrate. We demonstrate the fluidity of these condensates using light microscopy and address their network organization through electron microscopy, thereby bridging events to conclude on a liquid-liquid phase separation behavior. Additionally, we observe the recruitment of DnaB in the droplets, concomitant with the release of DciA. We show that the well-known helicase loader DnaC from Escherichia coli is also competent to form these phase-separated condensates in the presence of ssDNA. Our phenomenological data are still preliminary as regards the existence of these condensates in vivo, but open the way for exploring the potential involvement of DciA in the formation of non-membrane compartments within the bacterium to facilitate the assembly of replication players on chromosomal DNA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Microbial uricase is an essential enzyme in purine degradation and the development of low-purine food. High enzyme activity and an appropriate optimum pH must be established for low-purine food. Uricases from Neurospora crassa , Streptomyces coelicolor , Alkalihalobacillus clausii , Bacillus subtilis , and Brevibacterium casei were heterologously expressed in Escherichia coli . Uricase from Alkalihalobacillus clausii (AC-PUCL) showed the most potent enzyme activity (249.19 IU/mL) at 37 °C and pH 7.0. This is close to the pH of plant-based food. The K m and K m / K cat values of AC-PUCL were 30.12 μM and 1.46 s -1 μM -1 , respectively. Furthermore, the crystal structures of uricases from different sources revealed that hydrogen bonds could enhance substrate affinity and strong enzyme activity. In addition, the high enzyme activity may be contributed by the active pockets with an appropriate size. Finally, AC-PUCL helped reduce the purine substances in soybean, pea, and kidney bean, with residual uric acid can not be detected at pH 8.6, suggesting a promising industrial application.

Among all photosynthetic life forms, cyanobacteria exclusively possess a water-soluble, light-sensitive carotenoprotein complex known as orange carotenoid proteins (OCPs), crucial for their photoprotective mechanisms. These protein complexes exhibit both structural and functional modularity, with distinct C-terminal (CTD) and N-terminal domains (NTD) serving as light-responsive sensor and effector regions, respectively. The majority of cyanobacterial genomes contain genes for OCP homologs and related proteins, highlighting their essential role in survival of the organism over time. Cyanobacterial photoprotection primarily involves the translocation of carotenoid entity into the NTD, leading to remarkable conformational changes in both domains and formation of metastable OCP R . Subsequently, OCP R interacts with phycobiliprotein, inducing the quenching of excitation energy and a significant reduction in PS II fluorescence yield. In dark conditions, OCP R detaches from phycobilisomes and reverts to OCP O in the presence of fluorescent recovery proteins (FRP), sustaining a continuous cycle. Research suggests that the modular structure of the OCPs, coupled with its unique light-driven dissociation and re-association capability, opens avenues for exploiting its potential as light-controlled switches, offering various biotechnological applications., Competing Interests: Declarations. Conflict of interest: There are no known conflicts in financial interests or personal relationships that could have developed to influence the research work., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Oscillation of the active form of the initiator protein DnaA (ATP-DnaA) allows for the timely regulation for chromosome replication. After initiation, DnaA-bound ATP is hydrolyzed, producing inactive ADP-DnaA. For the next round of initiation, ADP-DnaA interacts with the chromosomal locus DARS2 bearing binding sites for DnaA, a DNA-bending protein IHF, and a transcription activator Fis. The IHF binding site is about equidistant between the DnaA and Fis binding sites within DARS2. The DARS2-IHF-Fis complex promotes ADP dissociation from DnaA and furnishes ATP-DnaA at the pre-initiation stage, which dissociates Fis in a negative-feedback manner. However, regulation for IHF binding as well as mechanistic roles of Fis and specific DNA structure at DARS2 remain largely unknown. We have discovered that negative DNA supercoiling of DARS2 is required for stimulating IHF binding and ADP dissociation from DnaA in vitro. Consistent with these, novobiocin, a DNA gyrase inhibitor, inhibits DARS2 function in vivo. Fis Gln68, an RNA polymerase-interaction site, is suggested to be required for interaction with DnaA and full DARS2 activation. Based on these and other results, we propose that DNA supercoiling activates DARS2 function by stimulating stable IHF binding and DNA loop formation, thereby directing specific Fis-DnaA interaction., (© The Author(s) 2025. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Halorhodospira (Hlr.) halophila strain BN9622 is an extremely halophilic and alkaliphilic purple phototrophic bacterium and has been widely used as a model for exploring the osmoadaptive and photosynthetic strategies employed by phototrophic extreme halophiles that enable them to thrive in hypersaline environments. Here we present the cryo-EM structures of (1) a unique native Hlr. halophila triple-complex formed from light-harvesting (LH1), the reaction center (RC), and high-potential iron-sulfur protein (HiPIP) at 2.44 Å resolution, and (2) a HiPIP-free LH1-RC complex at 2.64 Å resolution. Differing from the LH1 in the Hlr. halophila LH1-LH2 co-complex where LH1 encircles LH2, the RC-associated LH1 complex consists of 16 (rather than 18) αβ-subunits circularly surrounding the RC. These distinct forms of LH1 indicate that the number of subunits in a Hlr. halophila LH1 complex is flexible and its size is a function of the photocomplex it encircles. Like LH1 in the LH1-LH2 co-complex, the RC-associated LH1 complex also contained two forms of αβ-polypeptides and both dimeric and monomeric molecules of bacteriochlorophyll a. The majority of the isolated Hlr. halophila LH1-RC complexes contained the electron donor HiPIP bound to the surface of the RC cytochrome subunit near the heme-1 group. The bound HiPIP consisted of an N-terminal functional domain and a long C-terminal extension firmly attached to the cytochrome subunit. Despite overall highly negative surface-charge distributions for both the cytochrome subunit and HiPIP, the interface between the two proteins was relatively uncharged and neutral, forming a pathway for electron tunneling. The structure of the Hlr. halophila LH1-RC-HiPIP complex provides insights into the mechanism of light energy acquisition coupled with a long-distance electron donating process toward the charge separation site in a multi-extremophilic phototroph., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

The intricate process of 50S ribosomal subunit assembly in Bacillus subtilis involves multiple parallel pathways converging into a crucial intermediate known as the 45S particle. RbgA and YphC, play pivotal roles in completing the maturation of the functional sites in the 45S particle. In this work, we found that RbgA and YphC can independently bind the 45S particle with high affinity, but when RbgA binds first to the particle, it significantly increases the binding affinity of YphC. Using cryo-electron microscopy, we determined that the changes exerted by RbgA and YphC when binding independently closely resemble those observed when the two factors bind to the 45S particle simultaneously. However, the structural analysis revealed that RbgA binding causes a conformational change that uncovers the binding site for YphC, thus increasing its binding affinity. We concluded that the functional interplay between RbgA and YphC primarily revolves around one factor promoting the binding of the other, rather than the binding of the two factors inducing entirely new conformational changes compared with those induced by the factors individually. These results highlight the synergic mechanism between two essential assembly factors, underscoring the intricate mechanism bacteria use to maximize the efficiency of the ribosome assembly process., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Streptococcus pyogenes, or Group A Streptococcus (GAS), is a commensal bacteria and human pathogen. Central to GAS pathogenesis is the presence of prophage encoded virulence genes. The conserved phage gene for the protein paratox (Prx) is genetically linked to virulence genes, but the reason for this linkage is unknown. Prx inhibits GAS quorum sensing and natural competence by binding the transcription factor ComR. However, inhibiting ComR does not explain the virulence gene linkage. To address this, we took a mass spectrometry approach to search for other Prx interaction partners. The data demonstrates that Prx binds numerous DNA-binding proteins and transcriptional regulators. We show binding of Prx in vitro with the GAS protein Esub1 (SpyM3_0890) and the phage protein JM3 (SpyM3_1246). An Esub1:Prx complex X-ray crystal structure reveals that Esub1 and ComR possess a conserved Prx-binding helix. Computational modelling predicts that the Prx-binding helix is present in several, but not all, binding partners. Namely, JM3 lacks the Prx-binding helix. As Prx is conformationally dynamic, this suggests partner-dependent binding modes. Overall, Prx acts as a metabolic regulator of GAS to maintain the phage genome. As such, Prx maybe a direct contributor to the pathogenic conversion of GAS., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The pathway of bacterial cysteine biosynthesis is gaining traction for the development of antibiotic adjuvants. Bacterial cysteine biosynthesis is generally facilitated by two enzymes possessing O -acetyl-l-serine sulfhydrylases (OASS), CysK and CysM. In Staphylococcus aureus , there exists a single OASS homologue, Sa CysK. Knockout of Sa CysK was found to increase sensitivity to oxidative stress, making it a relevant target for inhibitor development. Sa CysK also forms two functional complexes via interaction with the preceding enzyme in the pathway serine acetyltransferase (CysE) or the transcriptional regulator of cysteine metabolism (CymR). These interactions occur through insertion of a C-terminal peptide of CysE or CymR into the active site of Sa CysK, inhibiting OASS activity, and therefore represent an excellent starting point for developing Sa CysK inhibitors. Here, we detail the characterization of CysE and CymR-derived C-terminal peptides as inhibitors of Sa CysK. Using a combination of X-ray crystallography, surface plasmon resonance, and enzyme inhibition assays, it was determined that the CymR-derived decapeptide forms extensive interactions with Sa CysK and acts as a potent inhibitor ( K D = 25 nM; IC 50 = 180 nM), making it a promising lead for the development of Sa CysK inhibitors. To understand the determinants of this high-affinity interaction, the structure-activity relationships of 16 rationally designed peptides were also investigated. This identified that the C-terminal pentapeptide of CymR facilitates the high-affinity interaction with Sa CysK and that subtle structural modification of the pentapeptide is possible without impacting potency. Ultimately, this work identified CymR pentapeptides as a promising scaffold for the development of antibiotic adjuvants targeting Sa CysK.

Streptococcus oligofermentans , a Gram-positive bacterium found in the oral microbiome, shows promise as an oral probiotic for preventing dental caries. It exhibits a reverse correlation with Streptococcus mutans , a key caries-causing pathogen, likely due to its production of hydrogen peroxide, a process mediated by quorum sensing (QS). In this work, we set out to develop novel lactam-based cyclic analogues of the competence stimulating peptide (CSP) signal utilized by S. oligofermentans for QS activation. To this end, we first conducted a ring position scan, where we determined the best positions within the CSP sequence to use for macrolactamization. We then conducted systematic ring size and bridge position scans to fine-tune the cyclic peptide conformation and identified a cyclic analogue, CSP-cyc(K2E2), with enhanced biological activity, 7-fold more active than the native CSP signal. This analogue also exhibited improved stability toward enzymatic degradation, demonstrating this analogue's potential utility as a chemical probe to study interspecies interactions between oral microbes and as a potential therapeutic agent. Overall, our lead cyclic analogue could be applied to augment the biotherapeutic potential of S. oligofermentans against S. mutans infections.

Phytochromes perceive subtle changes in the light environment and convert them into biological signals by photoconversion between the red-light absorbing (Pr) and the far-red-absorbing (Pfr) states. In the primitive bacteriophytochromes this includes refolding of a tongue-like hairpin loop close to the chromophore, one strand of an antiparallel β-sheet being replaced by an α-helix. However, the strand sequence in the cyanobacterial phytochrome Cph1 is different from that of previously investigated bacteriophytochromes and has a higher β-sheet propensity. We confirm here the transition experimentally and estimate minimum helix length using dynamic nuclear polarisation (DNP) magic angle spinning NMR. Sample conditions were optimized for protein DNP NMR studies at high field, yielding Boltzmann enhancements ϵ B of 19 at an NMR field of 18.801 T. Selective labelling of Trp, Ile, Arg, and Val residues with 13 C and 15 N enabled filtering for pairs of labelled amino acids by the 3D CANCOCA technique to identify signals of the motif 483 Ile-Val-Arg 485 (IVR) present in both sheet and helix. Those signals were assigned for the Pfr state of the protein. Based on the chemical shift pattern, we confirm for Cph1 the formation of a helix covering the IVR motif., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

d-Allulose 3-epimerase (DAEase) derived from Clostridium bolteae has excellent properties in the catalytic production of d-allulose, a rare sugar with unique biological functions. However, the industrial application of C. bolteae DAEase (Cb-DAEase) for d-allulose production is hindered by its low enzyme activity, poor long-term thermostability, and pH tolerance. In this study, we identified potential noncatalytic residues in Cb-DAEase using methods such as proline substitution, surface charge engineering, and surface residue prediction. The effects of these residues were experimentally validated, followed by structural analysis, which led to the generation of multisite mutants through cross-regional structural combinations. The obtained mutant Cb-R2P-E6P-D137C showed 155.6% of the enzyme activity of the wild type, and the K cat / K m increased by 1.3-fold, an elevated half-life of 15.7 min, and an elevated T m value of 1.1 °C. The mutant Cb-R2P-E6P-A83D-D137C had 139.7% of the enzyme activity of the wild type, the K cat / K m increased by 1.2-fold, with an elevated half-life of 12.3 min, an elevated T m value of 0.8 °C, and maintained 68% of the enzyme activity at pH 5.0. The findings outlined a feasible approach for the rational design of multiple preset functions of target enzymes simultaneously.

The homo-dodecameric ring-shaped trp RNA binding attenuation protein (TRAP) from Alkalihalobacillus halodurans (Aha) binds up to twelve tryptophan ligands (Trp) and becomes activated to bind a specific sequence in the 5' leader region of the trp operon mRNA, thereby downregulating biosynthesis of Trp. Thermodynamic measurements of Trp binding have revealed a range of cooperative behavior for different TRAP variants, even if the averaged apparent affinities for Trp have been found to be similar. Proximity between the ligand binding sites, and the ligand-coupled disorder-to-order transition has implicated nearest-neighbor interactions in cooperativity. To establish a solid basis for describing nearest-neighbor cooperativity in TRAP, we engineered variants constructed with two subunits connected by a flexible linker (dTRAP). We mutated the binding sites of alternating protomers such that only every other site was competent for Trp binding (WT-Mut dTRAP). Ligand binding monitored by NMR, calorimetry, and native mass spectrometry revealed strong cooperativity in dTRAP containing adjacent binding-competent sites, but a severe binding defect when the wild-type sites were separated by mutated sites. Cryo-EM experiments of dTRAP in its ligand-free apo state, and both dTRAP and WT-Mut dTRAP in the presence of Trp, revealed progressive stabilization of loops that gate the Trp binding site and participate in RNA binding. These studies provide important insights into the thermodynamic and structural basis for the observed ligand binding cooperativity in TRAP. Such insights can be useful for understanding allosteric control networks and for the development of those with defined ligand sensitivity and regulatory control., Competing Interests: Competing interests statement:The authors declare no competing interest.

Rotation of the bacterial flagellum, the first identified biological rotary machine, is driven by its stator units. Knowledge gained about the function of stator units has increasingly led to studies of rotary complexes in different cellular pathways. Here, we report that a tetrameric PilZ family protein, FlgX, is a structural component underneath the stator units in the flagellar motor of Campylobacter jejuni . FlgX forms a stable tetramer that does not bind cyclic di-GMP (c-di-GMP), unlike other canonical PilZ domain-containing proteins. Cryoelectron tomography and subtomogram averaging of flagellar motors in situ provide evidence that FlgX interacts with each stator unit and plays a critical role in stator ring assembly and stability. Furthermore, FlgX is conserved and was most likely present in the common ancestor of the phylum Campylobacterota . Overall, FlgX represents a divergence in function for PilZ superfamily proteins as well as a player in the key stator-rotor interaction of complex flagellar motors., Competing Interests: Competing interests statement:The authors declare no competing interest.

Many bacteria swim in liquid or swarm on surface using the flagellum rotated by a motor driven by specific ion flow. The motor consists of the rotor and stator, and the stator converts the energy of ion flow to mechanical rotation. However, the ion pathway and the mechanism of stator rotation coupled with specific ion flow are still obscure. Here, we determined the structures of the sodium-driven stator of Vibrio , namely PomAB, in the presence and absence of sodium ions and the structure with its specific inhibitor, phenamil, by cryo-electron microscopy. The structures and following functional analysis revealed the sodium ion pathway, the mechanism of ion selectivity, and the inhibition mechanism by phenamil. We propose a model of sodium ion flow coupled with the stator rotation based on the structures. This work provides insights into the molecular mechanisms of ion specificity and conversion of the electrochemical potential into mechanical functions., Competing Interests: Competing interests statement:The authors declare no competing interest.

PhoU proteins are negative regulators of the phosphate response, regulate virulence, and contribute to antibiotic resistance. Staphylococcus aureus has multiple genes encoding PhoU homologs that regulate persister formation and potentially virulence, but the molecular mechanisms of this regulation are not fully understood. We used a bacterial adenylate cyclase two-hybrid system to assess interactions between PhoU homologs and other proteins known to interact with PhoU from Escherichia coli. S. aureus PhoU (also referred to as PhoU1) interacted with PhoU itself; PitR (also referred to as PhoU2) interacted with PitR itself. We identified potential structural and dimerization models for S. aureus PhoU homologs. Dimerization was confirmed using size exclusion chromatography of purified proteins. These results highlight the complex nature of PhoU proteins. Further analysis may elucidate the potential mechanisms for regulating gene expression, persister formation, and virulence in S. aureus .IMPORTANCEPhoU proteins affect pathogenesis and persister formation in many bacterial species. This protein is essential for signaling environmental phosphate levels in Escherichia coli but is still not well characterized in many other pathogenic bacterial strains. This work identifies some similarities and key differences in Staphylococcus aureus PhoU homologs compared to E. coli PhoU, specifically, PhoU and PitR from S. aureus form homodimers but do not appear to interact with PhoR or phosphate transporter proteins., Competing Interests: The authors declare no conflict of interest.

Blastochloris (Blc.) tepida is a hot spring purple nonsulfur phototrophic bacterium that contains bacteriochlorophyll (BChl) b . Here, we present a 2.21 Å cryo-EM structure of the thermostable light-harvesting 1-reaction center (LH1-RC) complex from Blc. tepida . The LH1 ring comprises 16 circularly arranged αβγ-subunits plus one αβ-subunit that surround the RC complex composed of C-, H-, L-, and M-subunits. In a comparative study, the Blc. tepida LH1-RC showed numerous electrostatic and hydrophobic interactions both within the LH1 complex itself and between the LH1 and the RC complexes that are absent from the LH1-RC complex of its mesophilic counterpart, Blc. viridis . These additional interactions result in a tightly packed LH1-RC architecture with a reduced accessible surface area per volume that enhances the thermal stability of the Blc. tepida complex and allows the light reactions of photosynthesis to proceed at hot spring temperatures. Moreover, based on high-resolution structural information combined with spectroscopic evidence, the unique photosynthetic property of the Blc. tepida LH1-RC─absorption of energy-poor near-infrared light beyond 1000 nm─can be attributed to strong hydrogen-bonding interactions between the C3-acetyl C═O of the LH1 BChl b and two LH1 α-Trp residues, structural rigidity of the LH1, and the enhanced exciton coupling of the LH1 BChls of this thermophile.