Inhibition of cytochrome bd oxidase in Mycobacterium tuberculosis by benzothiazole amides.

Discovery of novel Cytochrome bd oxidase inhibitors against Mycobacterium tuberculosis.

Rhizobium etli, a nitrogen-fixing bacterium, grows both in symbiosis (with plants) and in free-living state. While most metabolic models focus on its symbiotic form, this study refined the existing iOR363 model to account for free-living growth. By addition of a biomass formation reaction followed by model curation growth was simulated using various N-sources (NH₃, NO₂, and NO₃). At fixed succinate uptake rate (4.16 mmol/gDWC/h), ammonia yielded the highest growth rate of 0.259 h ⁻ ¹. To represent free-living N-fixing R. etli, a novel two-member community-like model, consisting of both growing and differentiated non-growing N-fixing cells with ammonia exchange, was developed. The XFBA approach, based on community Flux Balance Analysis (cFBA), was formulated to maintain fixed abundances rather than assuming equal growth rates. With a non-growing:growing abundance ratio of 1:9 in community, N-fixation resulted in lower growth rate of 0.1933 h ⁻ ¹ due to the high energy demand of N₂ assimilation compared to ammonia. Sensitivity analysis revealed that increased abundance of N-fixing cells from 5 to 30% led to decreases of 10% in N2-fixation and 25% in growth rate of growing member. Furthermore, Principal Component Analysis identified oxidative phosphorylation, TCA cycle, and glycolysis as key pathways differentiating flux distributions across N-sources. At high uptake of oxygen, causing nitrogenase inactivity, cytochrome bd oxidase was activated to scavenge oxygen, though at the cost of lower growth rate (by 12% per mmol increase in O2 uptake/gDWC/h). This study provided a platform to obtain insights to free-living state of R. etli which may have applications for other diazotrophs.

To determine the population variability in dextromethorphan metabolism by cytochrome (CY) P450 2D15 (CYP2D15) in dogs. Healthy pet dogs were recruited from 2018 through 2024 from the Inland Pacific Northwest and phenotyped by orally administering the Program in Individualized Medicine cocktail, which contains dextromethorphan, a CYP2D15-specific probe drug. Glucuronidase-treated urine samples collected 6 hours after dosing were assayed for dextromethorphan and dextrorphan concentrations. Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan (DOR/DXM Log MRs) were calculated. Dogs were genotyped for 5 missense CYP2D15 variants. Univariate and multivariate statistical approaches were used to evaluate associations between DOR/DXM Log MRs and demographic variables. 105 dogs, including 34 mixed breeds and 71 dogs from 20 different owner-designated breeds, were enrolled and completed the study. There was a wide distribution of DOR/DXM Log MRs, from 0.97 to 2.76, representing a log unit range of 1.8 (63-fold variation DOR/DXM Log MRs). Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan were normally distributed and unimodal. The mean (± SD) DOR/DXM Log MR was 2.04 ± 0.37. Multiple linear regression analysis showed significant association (R2 = 0.16) between DOR/DXM Log MRs and dog breed for Golden Retrievers (2.26 ± 0.29; N = 23) and Pugs (1.47 ± 0.29; N = 3). Log-transformed metabolic ratios of dextrorphan divided by dextromethorphan were not associated with dog sex, age, weight, or genotype. There is substantial variability in DOR/DXM Log MR values among individuals, which can be partially attributed to differences between breeds. These findings predict high variability in the metabolism of drugs by CYP2D15 associated with differences between dog breeds.

The nontuberculous mycobacterium (NTM) Mycobacterium abscessus (Mab) has emerged as a global health concern due to its high intrinsic resistance toward antibiotics. The search for anti-NTM inhibitors requires novel well-characterized targets. The cytochrome bd (cyt-bd) oxidase, which serves as an alternate terminal oxidase in mycobacteria, is a chemically validated drug target in Mycobacterium tuberculosis (Mtb). However, no genetic, biochemical, or structural studies have been described for the Mab enzyme. Successful targeting of the Mab cyt-bd oxidase requires an in-depth understanding of its mechanistic and regulatory elements. Here, we generated a homology model of Mab cyt-bd, including the alternate menaquinol-binding pocket, the predicted oxygen channel, the proposed redox modulation site (C266-C285), and the salt bridge pair, keeping the cysteine residues in proximity. A heterologous system was developed for whole-cell functional studies to characterize the impact of mutations in these critical domains on enzyme activity. Mutating W9, E98, F103, or E263 to alanine inhibited the enzyme totally, underscoring their importance in menaquinol binding, oxygen reduction, and/or redox modulation. The Mab cyt-bd C285A mutant displayed a reduction in oxygen consumption and ATP formation, a phenomenon also presented for the Mtb C285A mutant. In summary, this study presents the first structural and biochemical characterization of Mab cyt-bd oxidase, providing insights into the importance of mechanistic and regulatory elements of the Mab enzyme in a whole-cell setup, which will be of relevance for the design of anti-NTM and antituberculosis hit molecules targeting this oxidase.

The reduction of oxygen to water is crucial to life under aerobic conditions. Cytochrome bd oxidases perform this reaction with a very high oxygen affinity. Members of this protein family are solely found in prokaryotes and some archaea playing an important role in bacterial virulence and antibiotic resistance. Here, we combine mutagenesis, electrocatalysis, nitric oxide binding and release experiments as well as FTIR spectroscopy to demonstrate that proton delivery to the active site is essentially rate limiting in Cyt bd-I electrocatalysis. D58 and D105 of subunit CydB are crucial residues in this proton path and communicate via a hydrogen bond network. Oxygen reduction depends on proton delivery to the active site, which also influences NO release.

The iron-containing porphyrin heme is of high interest for the food industry for the production of artificial meat as well as for medical applications. Recently, the biotechnological platform strain Corynebacterium glutamicum has emerged as a promising host for animal-free heme production. Beyond engineering of complex heme biosynthetic pathways, improving heme export offers significant yet untapped potential for enhancing production strains. In this study, a growth-coupled biosensor was designed to impose a selection pressure on the increased expression of the hrtBA operon encoding an ABC-type heme exporter in C. glutamicum. For this purpose, the promoter region of the growth-regulating genes pfkA (phosphofructokinase) and aceE (pyruvate dehydrogenase) was replaced with that of PhrtB, creating biosensor strains with a selection pressure for hrtBA activation. Resulting sensor strains were used for plate-based selections and for a repetitive batch f(luorescent)ALE using a fully automated laboratory platform. Genome sequencing of isolated clones featuring increased hrtBA expression revealed three distinct mutational hotspots: (i) chrS, (ii) chrA, and (iii) cydD. Mutations in the genes of the ChrSA two-component system, which regulates hrtBA in response to heme levels, were identified as a promising target to enhance export activity. Furthermore, causal mutations within cydD, encoding an ABC-transporter essential for cytochrome bd oxidase assembly, were confirmed by the construction of a deletion mutant. Reversely engineered strains showed strongly increased hrtBA expression as well as increased cellular heme levels. These results further support the proposed role of CydDC as a heme transporter in bacteria. Mutations identified in this study therefore underline the potential of biosensor-based growth coupling and provide promising engineering targets to improve microbial heme production.

PstA is a structurally conserved c-di-AMP-binding protein that is broadly present among Firmicutes bacteria. Furthermore, PstA binds c-di-AMP at high affinity and specificity, indicating an important role in the c-di-AMP signaling network. However, the molecular function of PstA remains elusive. Our findings reveal contrasting roles of PstA in β-lactam resistance depending on c-di-AMP-binding status. We also define physiological conditions for PstA function during aerobic growth. Future efforts can exploit these conditions to identify PstA interaction partners under β-lactam stress.

Hepatocyte nuclear factor 4 alpha antisense 1 (HNF4A-AS1) is a long noncoding RNA (lncRNA) gene physically located next to the transcription factor HNF4A gene in the human genome. Its transcription products have been reported to inhibit the progression of hepatocellular carcinoma (HCC) and negatively regulate the expression of cytochrome P450s (CYPs), including CYP1A2, 2B6, 2C9, 2C19, 2E1, and 3A4. By altering CYP expression, lncRNA HNF4A-AS1 also contributes to the susceptibility of drug-induced liver injury. Thus, HNF4A-AS1 lncRNA is a promising target for controlling HCC and modulating drug metabolism. However, HNF4A-AS1 has four annotated alternative transcripts in the human genome browsers, and it is unclear which transcripts the small interfering RNAs or small hairpin RNAs used in the previous studies are silenced and which transcripts should be used as the target. In this study, four annotated and two newly identified transcripts were confirmed. These six transcripts showed different expression levels in different liver disease conditions, including metabolic dysfunction-associated steatotic liver disease, alcohol-associated liver disease, and obesity. The expression patterns of all HNF4A-AS1 transcripts were further investigated in liver cell growth from human embryonic stem cells to matured hepatocyte-like cells, HepaRG differentiation, and exposure to rifampicin treatment. Several HNF4A-AS1 transcripts highly displayed correlations with these situations. In addition, some of the HNF4A-AS1 transcripts also showed a strong correlation with CYP3A4 during HepaRG maturation and rifampicin exposure. Our findings provide valuable insights into the specific roles of HNF4A-AS1 transcripts, paving the way for more targeted therapeutic strategies for liver diseases and drug metabolism. SIGNIFICANCE STATEMENT: This study explores the alternative transcripts of HNF4A-AS1, showing how their expression changes in different biological conditions, from various liver diseases to the growth and differentiation of hepatocytes and drug metabolism. The generated knowledge is essential for understanding the independent roles of different transcripts from the same lncRNA in different liver diseases and drug metabolism situations.

Discovered in the 1920s, cytochrome bd is a terminal oxidase that has received renewed attention as a drug target since its atomic structure was first determined in 2016. Only found in prokaryotes, we study it here as a drug target for Mycobacterium tuberculosis (Mtb). Most previous drug discovery efforts toward cytochrome bd have involved analogues of the canonical substrate quinone, known as Aurachin D. Here, we report six new cytochrome bd inhibitor scaffolds determined from a computational screen and confirmed on target activity through in vitro testing. These scaffolds provide new avenues for lead optimization toward Mtb therapeutics.

Cytochrome bd (cyt-bd) oxidase, one of the two terminal oxidases in the Mycobacterium tuberculosis (Mtb) oxidative phosphorylation pathway, plays an indispensable role in maintaining the functionality of the metabolic pathway under stressful conditions. However, the absence of this oxidase in eukaryotic cells allows researchers to select it as a potential drug target for the synthesis of anti-tubercular (anti-TB) molecules. Cyt-bd inhibitors have often been combined with cytochrome bcc/aa3 super-complex inhibitors in anti-TB drug regimens to achieve a desired bactericidal response. The functional redundancy between both the terminal oxidases is responsible for this. The cryo-EM structure of cyt-bd oxidase from Mtb (PDB ID: 7NKZ) further accelerated the research to identify its inhibitor. Herein, we have summarized the reported anti-TB cyt-bd inhibitors, insight into the rationale behind targeting cyt-bd oxidase, and an outline of the architecture of Mtb cyt-bd oxidase.

Buruli ulcer (BU) is a chronic necrotizing skin disease caused by Mycobacterium ulcerans. Historically, the disease was treated by surgical excision of the skin lesions, until an 8-week combination therapy of rifampicin and streptomycin was introduced in 2004. This treatment modality was effective and reduced recurrence rates. Rifampicin is the most efficacious antibiotic for the treatment of BU and, should rifampicin-resistant M.ulcerans strains emerge, there is currently no replacement for it. As for mycobacterial diseases in general, there is a pressing need for the development of novel, fast-acting drugs. Under market economy conditions, repurposing of new tuberculosis drug candidates is the most promising avenue for alternative BU treatments. Our drug repurposing activities have led to the identification of several actives against M.ulcerans. In particular, the cytochrome bc1 complex inhibitor telacebec (Q203) is a promising drug candidate for the treatment of BU in Africa and Australia. While an active cytochrome-bd oxidase bypass limits the potency of the cytochrome-bc1-specific inhibitor telacebec against M.tuberculosis, classical lineage M.ulcerans strains rely exclusively on cytochrome-bc1 to respire. Hence, telacebec is effective at nanomolar concentration against M.ulcerans, and a high treatment efficacy in an experimental mouse infection model indicates that treatment of BU could be substantially shortened and simplified by telacebec.

The selective reduction of oxygen to water is crucial to life and a central process in aerobic organisms. It is catalyzed by several different enzymes, including cytochrome bd oxidases that are solely present in prokaryotes, including several pathogens. In addition, these enzymes play a crucial role in protection against oxidative stress, in virulence, adaptability and antibiotics resistance. The reduction of O2 occurs at the high spin D-type heme in all cytochrome bd oxidases, that is also the binding site for several ligands from signaling processes, including NO, H2S and CO.Here we present the electrocatalytic study of the cytochrome bd I and bd II oxidases from Escherichia coli (1,2) as well on other related bd oxidases. Structural parameters that are crucial for the reactivity towards oxygen are analyzed. The pH dependency of the binding and release of NO, an important signaling factor is presented. The influence of mutants in the proton channel on the NO release is discussed.(1) Grauel, A. Kägi, J. Rasmussen, T. Makarchuk, I. Oppermann, S. Moumbock, A., Wohlwend, D., Müller, R., Melin, F., Günther, S., Hellwig, P., Böttcher, B., and Friedrich, T., ‘Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D’ (2021) Nat. Commun., 12:6498.(2) Nikolaev, A., Safarian, S., Thesseling, A., Wohlwend, D., Friedrich, T., Michel, H., Kusumoto, T., Sakamoto, J., Melin, F., Hellwig P. ‘Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms’ (2021) Biochim. Biophys. Acta 1862, 148436.

BackgroundAcetaminophen is a commonly used medication by pregnant women and is known to cross the placenta. However, little is known about the biological mechanisms that regulate acetaminophen in the developing offspring. Cytochrome 2E1 (CYP2E1) is the primary enzyme responsible for the conversion of acetaminophen to its toxic metabolite. Ex vivo studies have shown that the CYP2E1 gene expression in human fetal liver and placenta is largely controlled by DNA methylation (DNAm) at CpG sites located in the gene body of CYP2E1 at the 5’ end. To date, no population studies have examined the association between acetaminophen metabolite and fetal DNAm of CYP2E1 at birth.MethodsWe utilized data from the Boston Birth Cohort (BBC) which represents an urban, low-income, racially and ethnically diverse population in Boston, Massachusetts. Acetaminophen metabolites were measured in the cord plasma of newborns enrolled in BBC between 2003 and 2013 using liquid chromatography-tandem mass spectrometry. DNAm at 28 CpG sites of CYP2E1 was measured by Illumina Infinium MethylationEPIC BeadChip. We used linear regression to identify differentially methylated CpG sites and the “DiffVar” method to identify differences in methylation variation associated with the detection of acetaminophen, adjusting for cell heterogeneity and batch effects. The false discovery rate (FDR) was calculated to account for multiple comparisons.ResultsAmong the 570 newborns included in this study, 96 (17%) had detectable acetaminophen in cord plasma. We identified 7 differentially methylated CpGs (FDR < 0.05) associated with the detection of acetaminophen and additional 4 CpGs showing a difference in the variation of methylation (FDR < 0.05). These CpGs were all located in the gene body of CYP2E1 at the 5’ end and had a 3–6% lower average methylation level among participants with detectable acetaminophen compared to participants without. The CpG sites we identified overlap with previously identified DNase hypersensitivity and open chromatin regions in the ENCODE project, suggesting potential regulatory functions.ConclusionsIn a US birth cohort, we found detection of cord biomarkers of acetaminophen was associated with DNAm level of CYP2E1 in cord blood. Our findings suggest that DNA methylation of CYP2E1 may be an important regulator of acetaminophen levels in newborns.

Nanaerobes are anewly described class of microorganisms that usea unique cytochrome bd oxidase to achieve nanaerobicrespiration at <2 μM dissolved oxygen (∼1% of atmosphericoxygen) but are not viable above this value due to the lack of otherterminal oxidases. Although sharing an overlapping ecological nichewith methanogenic archaea, the role of nanaerobes in methanogenicsystems has not been studied so far. To explore their occurrence andsignificance, we re-analyzed published meta-omic datasets from animalrumina and waste-to-energy digesters, including conventional anaerobicdigesters and anaerobic digesters with ultra-low oxygenation. Resultsshow that animal rumina share broad similarities in the microbialcommunity and system performance with oxygenated digesters, ratherthan with conventional anaerobic digesters, implying that trace levelsof oxygen drive the efficient digestion in ruminants. The rumen systemserves as an ideal model for the newly named nanaerobic digestion,as it relies on the synergistic co-occurrence of nanaerobes and methanogensfor methane yield enhancement. The most abundant ruminal bacterialfamily Prevotellaceae contains many nanaerobes, whichperform not only anaerobic fermentation but also nanaerobic respirationusing cytochrome bd oxidase. These nanaerobes generallyaccompany hydrogenotrophic methanogens to constitute a thermodynamicallyand physiologically consistent framework for efficient methane generation.Our findings provide new insights into ruminal methane emissions andstrategies to enhance methane generation from biomass.

Complete genome sequence of Halomonas alkaliantarctica MSP3 isolated from marine sediment, Jeju Island.

Aurachins are farnesylated quinolone alkaloids of bacterial origin and excellent inhibitors of the respiratory chain in pro- and eukaryotes. Therefore, they have become important tool compounds for the investigation of electron transport processes and they also serve as lead structures for the development of antibacterial and antiprotozoal drugs. Especially aurachin D proved to be a valuable starting point for structure-activity relationship studies. Aurachin D is a selective inhibitor of the cytochrome bd oxidase, which has received increasing attention as a target for the treatment of infectious diseases caused by mycobacteria. Moreover, aurachin D possesses remarkable activities against Leishmania donovani, the causative agent of leishmaniasis. Aurachins are naturally produced by myxobacteria of the genus Stigmatella as well as by some Streptomyces and Rhodococcus strains. The recombinant production of these antibiotics turned out to be challenging due to their complex biosynthesis and their inherent toxicity. Recently, the biotechnological production of aurachin D was established in E. coli with a titer which is higher than previously reported from natural producer organisms.

Mitochondrial function in adipocyte is an important aspect in maintaining metabolic homeostasis. Our previous observation showed that circulating levels of adrenomedullin (ADM) and mRNA and protein for ADM in omental adipose tissue were higher in patients with gestational diabetes mellitus (GDM), and these alterations are accompanied by glucose and lipid metabolic dysregulation, but the impact of ADM on mitochondrial biogenesis and respiration in human adipocyte remain elusive. The present study demonstrated that: (1) Increasing doses of glucose and ADM inhibit human adipocyte mRNA expressions of mitochondrial DNA (mtDNA)-encoded subunits of electron transport chain, including nicotinamide adenine dinucleotide dehydrogenase (ND) 1 and 2, cytochrome (CYT) b, as well as ATPase 6; (2) ADM significantly increases human adipocyte mitochondrial reactive oxygen species generation and this increase is reversed by ADM antagonist, ADM22-52, but treatment with ADM does not significantly affect mitochondrial contents in the adipocytes; (3) Adipocyte basal and maximal oxygen consumption rate are dose-dependently suppressed by ADM, thus results in impaired mitochondrial respiratory capacity. We conclude that elevated ADM observed in diabetic pregnancy may be involved in glucose and lipid dysregulation through compromising adipocyte mitochondrial function, and blockade of ADM action may improve GDM-related glucose and adipose tissue dysfunction.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments

Cellobiose dehydrogenase (CDH) is capable of direct electron transfer (DET) on electrodes and is a promising redox enzyme for bioelectrochemical applications. Its unique two-domain structure makes the function of CDH adsorbed on the surface of the electrode deeply affected by the external environment, such as ion species, strength, pH, and surface charge density. To date, however, the exact mechanism of how the external environment tailors the structure and dynamics of CDH adsorbed on the electrode surface still remains poorly understood. Here, multiscale simulations were performed to look for insight into the effect of Na+ and Ca2+ ions on the activation of CDH on oppositely charged self-assembled monolayer (NH2-SAM and COOH-SAM) surfaces with different surface charge densities (SCDs). Both Na+ and Ca2+ can promote CDH conformation switch from the open state to the closed state, while the promotion effect of Ca2+ is stronger than that of Na+ at the same conditions. However, the high ionic strength (IS) of Ca2+ renders the cytochrome (CYT) domain of CDH away from the NH2-SAM with low SCD. In contrast, whatever the IS, the NH2-SAM surface with high SCD can not only enhance the CYT-surface interaction but also achieve a closed-state conformation due to a similar role of Ca2+. Overall, this study gains molecular-level insights into the role of ion species and surface charge in modulating the structure and conformation of CDH on the SAM surface, thereby tailoring its activity.