This study focuses on the quaternary structure of the viper-secreted phospholipase A (PLA), a central toxin in viper envenomation. PLA enzymes catalyze the hydrolysis of the sn-2 ester bond of membrane phospholipids. Small-molecule inhibitors that act as snakebite antidotes, such as varespladib, are currently in clinical trials. These inhibitors likely bind to the enzyme in the aqueous cytosol prior to membrane-binding. Thus, understanding its controversial solution structure is key for drug design. Crystal structures of PLA in the PDB show at least four different dimeric conformations, the most well-known being "extended" and "compact". This variability among enzymes with >50 % sequence identity raises questions about their transferability to aqueous solution. Therefore, we performed extensive molecular dynamics (MD) simulations of several PLA enzymes in water to determine their quaternary structure under physiological conditions. The MD simulations strongly indicate that PLA enzymes adopt a "semi-compact" conformation in cytosol, a hybrid between extended and compact conformations. To our knowledge, this is the first study that determines the most favorable dimeric conformation of PLA enzymes in solution, providing a basis for advancements in snakebite envenoming treatment. Recognizing snakebite envenoming as a neglected tropical disease has driven the search for efficient, affordable alternatives to the current antivenoms. Therefore, understanding the main drug targets within snake venom is crucial to this achievement.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are involved in many cellular processes and possess unequalled catalytic versatility. Rational design through site-directed mutagenesis is a powerful strategy for creating tailor-made enzymes for a wide range of biocatalytic applications. PLP-dependent methionine γ-lyase (MGL), which degrades sulfur-containing amino acids, is an encouraging enzyme for many therapeutic purposes - from combating bacterial resistant strains and fungi to antitumor activity. A two-component biosystem MGL/S-alk(en)yl-l-cysteine sulfoxide (an uncommon substrate for this enzyme) produces antimicrobial thiosulfinates during the β-elimination reaction. SH-groups of the enzyme are modified by the products of this reaction, which leads to the inactivation of the enzyme. Successful and efficient rational stabilization of MGL from Clostridium novyi can be achieved using site-directed mutagenesis. We have managed to obtain an improved version of the enzyme better than the natural one regarding the β-elimination reaction. Cys118, Cys184 and Cys273 of MGL from Clostridium novyi were substituted by His and two Ala, respectively. The resulting Cys-del variant had 2-3-fold improved k/K value for conventional β-eliminating substrates and up to 10-fold increase in catalytic efficiency with S-substituted-l-cysteine sulfoxides compared to the wild-type MGL. The Cys-del MGL remained active under the thiosulfinates, which are products of β-elimination reaction of S-alk(en)yl-l-cysteine sulfoxides. Moreover, Cys-del variant proved to be more stable during shelf storage. Thus, we have created an effective enzyme component of the biocatalytic system that is capable of generating antimicrobial drugs - thiosulfinates.

Endometriosis affects about 10% of women of reproductive age, leading to a disabling gynecologic condition. Chronic pain, inflammation, and oxidative stress have been identified as the molecular pathways involved in the progression of this disease, although its precise etiology remains uncertain. Although mitochondria are considered crucial organelles for cellular activity, their dysfunction has been linked to the development of this disease. The purpose of this review is to examine the functioning of the mitochondrion in endometriosis: in particular, we focused on the mitochondrial dynamics of biogenesis, fusion, and fission. Since excessive mitochondrial activity is reported to affect cell proliferation, we also considered mitophagy as a mechanism involved in limiting disease development. To better understand mitochondrial activity, we also considered alterations in circadian rhythms, the gut microbiome, and estrogen receptors: indeed, these mechanisms are also involved in the development of endometriosis. In addition, we focused on recent research about the impact of numerous substances on mitochondrial activity; some of them may offer a future breakthrough in endometriosis treatment by acting on mitochondria and inhibiting cell proliferation.

Microorganisms play a crucial role in the degradation of microcystins (MCs), with most MC-degrading bacteria utilizing the mlr gene cluster (mlrABCD) mechanism. While previous studies have advanced our understanding of the structure, function, and degradation mechanisms of MlrA, MlrB, and MlrC, research on MlrD remains limited. Consequently, the molecular structure and specific catalytic processes of MlrD are still unclear. This study investigates MlrD from Sphingopyxis sp. USTB-05, utilizing bioinformatics tools for analysis and prediction, conducting homology analysis, and constructing the molecular structure of MlrD. Bioinformatics analysis suggests that MlrD is an alkaline, hydrophobic protein with good thermal stability and is likely located in the cell membrane as a membrane protein without a signal peptide. Homology analysis indicates that MlrD belongs to the PTR2 protein family and contains a PTR2 domain. Phylogenetic analysis reveals that MlrD follows both vertical and horizontal genetic transfer patterns during evolution. Homology modeling demonstrates that the three-dimensional structure of MlrD is primarily composed of 12 α-helices, with conserved residues between the N-terminal and C-terminal domains forming a large reaction cavity. This research broadens current knowledge of MC biodegradation and offers a promising foundation for future studies.

Macrolactin A (McA) is a secondary metabolite produced by Bacillus species. It has been known for its antimicrobial properties since the late 1980s, although the exact mechanism of its antibacterial activity remains unknown. In this study, we have found that McA is an inhibitor of protein synthesis in bacteria. Our conclusion is based on the results obtained by in vivo and in vitro bioreporter systems. We demonstrated that the inhibitory activity of McA is independent of bacterial species. However, the concentration of McA required to inhibit protein synthesis in the E. coli cell-free translational model was found to be 50 times lower than the concentration required in the S. aureus cell-free translational model. To investigate the mechanism of McA's inhibitory activity, we conducted a toe-printing assay, sequenced and annotated the genomes of McA-resistant Bacillus pumilus McA and its parental strain. The results showed that McA inhibits the initial step of the elongation phase of protein synthesis. We identified single and multiple nucleotide polymorphisms in the gene encoding the translation elongation factor Tu (EF-Tu). Molecular modeling showed that the McA molecule can form non-covalent bonds with amino acids at the interface of domains 1 and 2 of EF-Tu. A cross-resistance assay was conducted using kirromycin on B. pumilus McA. The results confirmed the assumption that McA has a mode of action similar to that of other elfamycin-like antibiotics (targeting EF-Tu). Overall, our study addresses a significant gap in our understanding of the mechanism of action of McA, a representative member of the macrolide antibiotics.

Obesity treatment requires an individualized approach, emphasizing the need to identify metabolic pathways of diagnostic relevance. Toll-like receptors (TLRs), particularly TLR2 and TLR4, play a crucial role in metabolic disorders, as receptor deficiencies improves insulin sensitivity and reduces obesity-related inflammation. Additionally, hydrogen sulfide (HS) influences lipolysis, adipogenesis, and adipose tissue browning through persulfidation. This study investigates the impact of a high-fat diet (HFD) on low molecular weight sulfur compounds in the visceral adipose tissue (VAT) of C57BL/6 and TLR2-deficient mice. It focuses on key enzymes involved in HS metabolism: cystathionine beta-synthase (CBS), cystathionine gamma-lyase (CGL), 3-mercaptopyruvate sulfurtransferase (MPST), and thiosulfate sulfurtransferase (TST). In C57BL/6 mice on HFD, MPST activity decreased, while CBS level increased, potentially compensating for HS production. In contrast, TLR2-deficient mice on HFD exhibited higher MPST activity but reduced level of CBS and CGL activity, suggesting that TLR2 deficiency mitigates HFD-induced changes in sulfur metabolism. TST activity was lower in TLR2-deficient mice, indicating an independent regulatory role of TLR2 in TST activity. Elevated oxidative stress, reflected by increased glutathione levels, was observed in wild-type mice. Interestingly, cysteine and cystine were detectable only in the VAT of the C57BL/6 ND group and were absent in all other groups. The capacity for hydrogen sulfide production in tissues from TLR2-/-B6 HFD group was significantly lower than in the C57BL/6 HFD group. In conclusion, TLR2 modulates sulfur metabolism, oxidative stress, and inflammation in obesity. TLR2 deficiency disrupts HS production and redox balance, potentially contributing to metabolic dysfunction, highlighting TLR2 as a potential therapeutic target for obesity-related metabolic disorders.

The parasite of the genus Leishmania is the causative agent of diseases that affect humans called leishmaniasis. These diseases affect millions of people worldwide and the currently existing drugs are either very toxic or the parasites acquire resistance. Therefore, new elimination mechanisms need to be elucidated so that new therapeutic strategies can be developed. Much has already been discussed about the role of neutrophils in Leishmania infection, and their participation is still controversial. A recent study showed that receptors present in the neutrophil membrane, the purinergic receptors, can control the infection when activated, but the triggering mechanism has not been elucidated. In this review, we will address the possible participation of purinergic receptors expressed in the neutrophil extracellular membrane that may be participating in the detection of Leishmania infection and their possible effects during parasitism.

Oxidative stress arises from an imbalance between reactive species (RS) production and the antioxidant defense, increasing the brain susceptibility to neurodegenerative and psychiatric diseases. Besides, changes in the expression or activity of neurotransmitter metabolism enzymes, such as monoamine oxidases (MAO), are also associated with mental disorders, including depression. Considering this, antioxidant and MAO-A activity inhibitory potential of six 2,3-chalcogenodihydrobenzofurans (2,3-DHBF) was investigated through in vitro and in silico tests. Compounds 1 to 5 incorporate sulfur (S) as chalcogen, whereas compound 6 integrates tellurium (Te). A screening (compounds 1-6) of cerebral MAO-A activity showed inhibitory activity for the compounds 2, 4, 5, and 6. Among sulfur compounds, compound 2 demonstrated superior scores in docking studies, yielding a value of - 9.9 kcal/mol. Selected for concentration-response curves, compounds 2 (with S) and 6 (with Te) inhibited MAO-A at concentrations equal to or higher than 25 μM. In a redox screening test, only compound 6 showed antioxidant effects. Concentration-response curves indicated that compound 6 reduced lipid peroxidation and protein carbonylation levels in mouse brain tissue (≥ 0.5 μM), as well as reduced RS levels (≥ 1 μM). Furthermore, the compound 6 (≥ 5 μM) was effective in reducing the ferric ion (FRAP). In radical scavenging tests such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), compound 6 showed significant results in concentrations from 50 μM and mimicked the enzyme glutathione S-transferase (GST) at 100 μM. In summary, this study demonstrated the cerebral antioxidant and/or MAO-A inhibition properties of 2,3-DHBF, presenting potential as neuroprotective candidates.

PA-BJ is a serine protease present in Bothrops jararaca venom that triggers platelet aggregation and granule secretion by activating the protease-activated receptors PAR-1 and PAR-4, without clotting fibrinogen. These receptors also have a relevant role in endothelial cells, however, the interaction of PA-BJ with other membrane-bound or soluble targets is not known. Here we explored the activity of PA-BJ on endothelial cell receptor, cytoskeleton, and coagulation proteins in vitro, and show the degradation of fibrinogen and protein C, and the limited proteolysis of actin, EPCR, PAR-1, and thrombomodulin. Antithrombin, factors XI and XIII and protein S were not cleaved by PA-BJ. Moreover, using surface plasmon resonance PA-BJ was demonstrated to bind to actin, EPCR, fibrinogen, PAR-1, and thrombomodulin, with dissociation constants (K) in the micromolar range. Considering that these proteins play critical roles in pathways of blood coagulation and maintenance of endothelium integrity, their binding and cleavage by PA-BJ could contribute to the alterations in hemostasis and cell permeability observed in B. jararaca envenomation process.

Anencephaly, the most severe type of neural tube defects (NTDs) in humans, occurs between the third and fourth gestational weeks (GW), involves the cranial part of the NT and results in the absence of the forebrain and skull. Exposed to amniotic fluid toxicity, neural tissue is degraded and prevented from development. Currently, little is known about the molecular bases of the disease and the possible involvement of glycans. In this context, considering the role played by gangliosides (GGs) in fetal brain development and the previous achievements of ion mobility separation (IMS) mass spectrometry (MS) in biomarker discovery, we report here on the introduction of this advanced analytical technique in NTD research, and its optimization for a comprehensive determination of anencephaly gangliosidome. Three native GG extracts from residual brains of anencephalic fetuses in 28, 35 and 37 GW were comparatively profiled by IMS MS, structurally analyzed by IMS MS/MS, and finally assessed against a native GG mixture from normal fetal brain. IMS MS provided data on 343 anencephaly gangliosides vs. only 157 known before and revealed for the first time the incidence of the entire penta- to octasialylated series. The comparative assay disclosed variations in GG expression with fetal age and a correlation of the pattern with the developmental stage. In contrast to the normal fetal brain, the neural tissue in anencephaly was found to contain an elevated number of polysialogangliosides and a lower expression of O-Ac- and GalNAc-modified glycoforms. These species worth further detailed investigation as new potential anencephaly markers.

Fibrinolytic enzymes are promising in treating cardiovascular diseases due to their capacity to dissolve blood clots. The fibrinolytic enzyme from Arthrospira platensis (FEAP) was purified by ion exchange chromatography to investigate its ability to activate plasminogen, as well as its thrombolytic and fibrinogenolytic potential. Subsequently, two different cytotoxic assays (MTT and NR) and hemolysis test were performed to evaluate FEAP's safety. Furthermore, cell migration and the genotoxic and hemolytic potential were also investigated. The purified enzyme showed thrombus degradation of 43 % and thrombolytic action directly on fibrin, which can reduce possible side effects, such as hemorrhage. MTT assay was more sensitive to determine the enzyme cytotoxicity, which decreased the viability of breast cancer tumor cells (Sarcoma-180 and MDA-MB-231) and macrophages (J774A.1). In addition, the enzyme also exhibited non-hemolytic, antimetastatic, and non-genotoxic characteristics. These findings are innovative for a fibrinolytic protease and may indicate that it is safe for people undergoing cancer treatment, reducing side effects such as hemorrhage, in addition to inhibiting tumor cells and preventing metastasis, which can help with chemotherapy treatment.

BAG3 is a universal adapter protein involved in various cellular processes, including the regulation of apoptosis, chaperone-assisted selective autophagy, and heat shock protein function. The interaction between small heat shock proteins (sHsps) and their α-crystallin domains (Acds) with full-length BAG3 protein and its IPV domain was analyzed using size-exclusion chromatography, native gel electrophoresis, and chemical cross-linking. HspB7 and the 3D mutant of HspB1 (which mimics phosphorylation) showed no interaction, HspB6 weakly interacted, and HspB8 strongly interacted with full-length BAG3. In contrast to the full-length sHsps, their α-crystallin domains (AcdB1, AcdB5, and AcdB6) were able to interact with BAG3, with AcdB8 again being the strongest interactor. Among all the full-length sHsps analyzed, only HspB8 bound to the IPV domain of BAG3. AcdB1, AcdB5, AcdB6, and AcdB8 interacted with the IPV domain of BAG3, with AcdB8 displaying the highest binding efficiency. The stoichiometry of crosslinked complexes formed by HspB8 (or its Acd) and the IPV domain of BAG3 was 2:1, whereas for the other sHsps and their Acds, it was 1:1. These findings suggest that while the IPV domain of BAG3 and the Acds of sHsps play an important role in binding, other structural regions significantly contribute to this interaction. The unique binding efficiency between BAG3 and HspB8 may be attributed to the intrinsic disorder and simple oligomeric structure of HspB8.

Fatty acid desaturases (FADs) belong to of the oxygenase superfamily. They play important roles in metabolic pathways and adaption mechanisms in a wide range of organisms, including bacteria and humans. These enzymes dehydrogenate a single bond in the acyl chain of fatty acids (FAs), forming a double bond. Multiple parameters influence the precise position of double bond formation and acyl chain docking in the catalytic pocket of various FADs, such as the length of an acyl chain, the position of previously generated double bonds, the location of the enzyme's metal catalytic site, and so on. The "counting" mode differs from one FAD to another. The cyanobacterium Synechocystis sp. strain PCC 6803 has four FADs (Δ9, Δ12, Δ6, and Δ15 or ω3) that synthesize mono-, di-, tri-, and tetraenoic FAs. The substrate preferences and "counting" modes for the first three FADs have been identified, but the substrate specificity for the terminal ω3-FAD remains uncertain. We used molecular cloning, heterologous expression with exogenous FAs, and molecular docking to explore the substrate selectivity and counting mode of ω3-FAD. Our results show that ω3-FAD "counts" from the carboxyl (Δ) end, introduces a double bond between 15 and 16 atoms, and requires a specific acyl substrate configuration with two pre-existing double bonds at Δ and Δ positions.

Staphylocoagulase (Coa) plays a critical role in the pathogenicity of Staphylococcus aureus (S. aureus). The present study was undertaken to investigate the underlying mechanism which helicid (HEL) suppressed the virulence factor Coa, as well as to assess the synergistic inhibitory effects of HEL in conjunction with antibiotics, thereby establishing the potential of HEL as an antibacterial adjuvant. We employed coagulation and biofilm assays to comprehensively assess the inhibitory impact of HEL on S. aureus pathogenicity. The thermal shift assay demonstrated that HEL exerted a direct impact on the protein stability of Coa, evidenced by a 6 °C change in melting temperature (ΔTm) at a concentration of 100 μM. HEL binding to Coa proteins was further validated by molecular dynamics simulations and fluorescence quenching. Molecular docking and point mutation assays identified S23 and D112 as crucial binding sites for HEL and Coa. Furthermore, HEL has been observed to potentiate the bactericidal properties of ceftaroline fosamil (CEF-F), concurrently diminishing the resistance exhibited by S. aureus towards CEF-F, as demonstrated by antibiotic synergy tests and resistance induction assays. The combination of HEL and CEF-F effectively reduced the number of bacteria and improved the survival of both Galleria mellonella larvae and mice. Additionally, a significant decrease was observed in the levels of TNF-α, IL-6, and IFN-γ in mice broncho-alveolar lavage fluid (BALF). Ultimately, our findings confirmed that the direct binding of HEL to Coa could diminish the pathogenicity of S. aureus. Moreover, the combination with CEF-F substantially reduced the lethality associated with S. aureus-infected pneumonia and extended the efficacy of the antibiotic.

This mini review focuses on the phenomenon of homeoviscous adaptation (HVA). The concept, which dominated for decades, had a significant impact on membrane and lipid research. It includes the functional characterization of biological membranes and their domains, the role of lipids and fatty acids in cell metabolic control, and the characterization of fatty acid desaturases and their roles in membrane properties modulation. This hypothesis led to the discovery of a feed-back manner of desaturase expression and membrane-associated temperature sensors in bacteria.

Haemocyanin-derived peptides were previously found in semi-purified fractions of mucus secretion from the snail Achatina fulica, which exhibited an inhibitory effect on Staphylococcus aureus strains. Here, an in silico rational design strategy was employed to generate new antimicrobial peptides (AMPs) from A. fulica haemocyanin-derived peptides (AfH). The designed peptides were chemically synthetized using the Fmoc strategy, and their antimicrobial activity against Escherichia coli and S. aureus strains was evaluated using the broth microdilution method. In addition, the cytotoxic activity on Vero, HaCat, and human erythrocyte cells was also determined. The results demonstrated that 15-residue alpha-helical and cationic synthetic peptides exhibited the highest biological activity against Gram-positive strains, with minimum inhibitory concentrations (MIC) in the range from 7.5 to 30 μM. The positive selectivity index suggests a higher selectivity, primarily on the microorganisms evaluated, but not on eukaryotic cells. In this study, A. fulica hemocyanin was identified as an appropriate protein model for the rational design of AMPs against bacteria of public health significance. Further studies are required to evaluate the activity of the peptides on Gram-negative bacteria other than E. coli.

Thiadiazines are heterocyclic compounds known for some pharmacological activities. However, the ability of these compounds and their derivatives to act as antibacterial agents and inhibitors of the efflux system in resistant bacteria remains unknown. This study aims to evaluate the antibacterial and NorA efflux pump inhibitory activities of thiadiazine-derived compounds (IJ14, IJ15, IJ16, IJ17, IJ18, IJ19, and IJ20) against the Staphylococcus aureus 1199B strain. Minimum Inhibitory Concentration (MIC) tests and antibacterial activity assessment through NorA efflux system inhibition were performed using microdilution assays in 96-well plates. Additionally, ethidium bromide (EtBr) fluorescence emission assays were conducted to evaluate efflux system inhibition. The methodology revealed that the IJ17 and IJ20 compounds presented MIC values of 256 and 597.3 μg/mL, respectively. The efflux pump inhibition assessment using the microdilution method showed significant results for all compounds, which also increased the fluorescence rates emitted by EtBr. Consequently, thiadiazine-derived compounds exhibit promising results in targeting a key bacterial resistance mechanism, underscoring the need for further studies, such as molecular tests, to evaluate their mechanism of action and clarify the feasibility and efficacy of these compounds as antibacterial agents.

Frataxin plays vital roles in various iron related processes. Arabidopsis thaliana FRATAXIN HOMOLOG (AtFH) is the first identified plant frataxin and has been found to regulate the last step of heme biosynthesis. Here, we report the involvement of AtFH in heme catabolism by regulating the activity of heme oxygenase. AtFH forms a homodimer, and its crystal structure shows the dimeric interactions. A structural comparison with known frataxin structures suggests the iron binding sites, and the site for heme oxygenase activity is possibly located in a region containing Glu78. The results indicate a previously uncharacterized role of plant frataxin in heme catabolism.

Skeletal muscle has an important role in whole body energy metabolism and various proteases are involved in skeletal muscle functions. We have previously identified the cysteine protease legumain in cultured human skeletal muscle cells. However, the potential role of legumain in regulation of energy metabolism remains unexplored. This study aimed to investigate cellular uptake, processing, and activation of prolegumain in human myotubes. Additionally, we sought to determine the effects of prolegumain on energy substrate metabolism in these cells. During differentiation of human myoblast to myotubes, legumain mRNA expression and activity were upregulated. Interestingly, legumain activity in myotubes was inversely correlated with the body mass index (BMI) of the obese cell donors. Myotubes exposed to conditioned medium enriched in prolegumain during the last two days of differentiation demonstrated the capacity to internalize and process prolegumain into its active form. Pre-treatment with prolegumain induced a metabolic shift towards increased fatty acid uptake in myotubes, as evidenced by elevated oleic acid uptake whereas glucose uptake and oxidation were reduced. The metabolic changes were not reversed by a legumain inhibitor, indicating a different mechanism for this effect. The metabolic alterations were accompanied by increased mRNA expression of the fatty acid transporter CD36, whereas the glucose transporter GLUT1 mRNA level remained unchanged. These findings suggest that legumain may play a regulatory role in skeletal muscle energy metabolism, highlighting its potential as a novel therapeutic target of metabolic disorders.

Bacterial methionine biosynthesis is an attractive target for research due to its central role in cellular metabolism, as most steps of this pathway are missing in mammals. Up to now little is known about sulfur metabolism in pathogenic Clostridia species, making the study of the enzymes of Cys/Met metabolism in Clostridium tetani particularly relevant. Analysis of the C. tetani genome has shown that the bacterium is capable of synthesizing methionine by direct sulfhydration. In this study, we describe purification of recombinant O-acetylhomoserine sulfhydrylase, a member of the Cys/Met metabolism pyridoxal 5'-phosphate-dependent enzyme family, from C. tetani for the first time. The gene encoding O-acetylhomoserine sulfhydrylase was cloned into the pET-28a(+) vector and expressed in Escherichia coli. The expression product was purified and identified as a 462-amino acid protein with a molecular mass of ∼50 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The deduced amino acid sequence of the C. tetani enzyme showed a high degree of similarity to O-acetylhomoserine sulfhydrylases from other bacterial sources. We confirmed the O-acetylhomoserine sulfhydrylase activity, and found the enzyme to be optimally active at pH 7.5 and 50 °C. The native enzyme assembles into a homotetramer of approx. 200 kDa as revealed by gel filtration. The obtained enzyme is capable of l-methionine formation using methanethiol as a sulfur source, that has been revealed by H NMR spectral data. These findings broaden the understanding of the role of O-acetylhomoserine sulfhydrylase in C. tetani Cys/Met metabolism and provide a basis for its future investigations and research.

Kingella kingae, an emerging pediatric pathogen, secretes the pore-forming toxin RtxA, which has been implicated in the development of various invasive infections. RtxA is synthesized as a protoxin (proRtxA), which gains its biological activity by fatty acylation of two lysine residues (K558 and K689) by the acyltransferase RtxC. The low acylation level of RtxA at K558 (2-23 %) suggests that the complete acylation at K689 is crucial for toxin activity. Using a bacterial two-hybrid system, we show that substitutions of K558, but not K689, partially reduce the interaction of proRtxA with RtxC and that the acyltransferase interacts independently with each acylated site in vivo. While substitutions of K558 had no effect on the acylation of K689, substitutions of K689 resulted in an average 40 % increase in the acylation of K558. RtxA mutants monoacylated at either K558 or K689 irreversibly bound to erythrocyte membranes, with binding efficiency corresponding to the extent of lysine acylation. However, these mutants lysed erythrocytes with similarly low efficiency as nonacylated proRtxA and showed only residual overall membrane activity in planar lipid bilayers. Interestingly, despite forming fewer pores, the monoacylated mutants exhibited single-pore characteristics, such as conductance and lifetime, similar to those of intact RtxA. These findings indicate that the acylation at either K558 or K689 is sufficient for the irreversible insertion of RtxA into the membrane, but not for the efficient formation of membrane pores. Alternatively, K558 and K689 per se may play a crucial structural role in pore formation, regardless of their acylation status.