Depolymerization of the polyester-polyurethane by amidase GatA250 and enhancing the production of 4,4'-methylenedianiline with cutinase LCC.

Polyurethane (PU) is a complex polymer synthesized from polyols and isocyanates. It contains urethane bonds that resist hydrolysis, which decreases the efficiency of biodegradation. In this study, we first expressed the amidase GatA250, and then, assessed the enzymatic characterization of GatA250 and its efficiency in degrading the polyester-PU. GatA250 degraded self-synthesized thermoplastic PU film and postconsumption foam with degradation efficiency of 8.17% and 4.29%, respectively. During the degradation, the film released 14.8 µm 4,4'-methylenedianiline (MDA), but 1,4-butanediol (BDO) and adipic acid (AA) were not released. Our findings indicated that GatA250 only cleaved urethane bonds in PU, and the degradation efficiency was extremely low. Hence, we introduced the cutinase LCC, which possesses hydrolytic activity on the ester bonds in PU, and then used both enzymes simultaneously to degrade the polyester-PU. The combined system (LCC-GatA250) had higher degradation efficiency for the degradation of PU film (42.2%) and foam (13.94%). The combined system also showed a 1.80 time increase in the production of the monomer MDA, and a 1.23 and 3.62 times increase in the production of AA and BDO, respectively, compared to their production recorded after treatment with only GatA250 or LCC. This study provides valuable insights into PU pollution control and also proposes applicable solutions to manage PU wastes through bio-recycling.

Dual function of LapB (YciM) in regulating Escherichia coli lipopolysaccharide synthesis.

Levels of lipopolysaccharide (LPS), an essential glycolipid on the surface of most gram-negative bacteria, are tightly controlled-making LPS synthesis a promising target for developing new antibiotics. Escherichia coli adaptor protein LapB (YciM) plays an important role in regulating LPS synthesis by promoting degradation of LpxC, a deacetylase that catalyzes the first committed step in LPS synthesis. Under conditions where LPS is abundant, LapB recruits LpxC to the AAA+ protease FtsH for degradation. LapB achieves this by simultaneously interacting with FtsH through its transmembrane helix and LpxC through its cytoplasmic domain. Here, we describe a cryo-EM structure of the complex formed between LpxC and the cytoplasmic domain of LapB (LapBcyto). The structure reveals how LapB exploits both its tetratricopeptide repeat (TPR) motifs and rubredoxin domain to interact with LpxC. Through both in vitro and in vivo analysis, we show that mutations at the LapBcyto/LpxC interface prevent LpxC degradation. Unexpectedly, binding to LapBcyto also inhibits the enzymatic activity of LpxC through allosteric effects reminiscent of LpxC activation by MurA in Pseudomonas aeruginosa. Our findings argue that LapB regulates LPS synthesis in two steps: In the first step, LapB inhibits the activity of LpxC, and in the second step, it commits LpxC to degradation by FtsH.

Toward More Selective Antibiotic Inhibitors: A Structural View of the Complexed Binding Pocket of E. coli Peptide Deformylase.

Peptide deformylase (PDF) is involved in bacterial protein maturation processes. Originating from the interest in a new antibiotic, tremendous effort was put into the refinement of PDF inhibitors (PDFIs) and their selectivity. We obtained a full NMR backbone assignment the emergent additional protein backbone resonances of ecPDF 1-147 in complex with 2-(5-bromo-1H-indol-3-yl)-N-hydroxyacetamide (2), a potential new structural scaffold for more selective PDFIs. We also determined the complex crystal structures of E. coli PDF (ecPDF fl) and 2. Our structure suggests an alternative ligand conformation within the protein, a possible starting point for further selectivity optimization. The orientation of the second ligand conformation in the crystal structure points toward a small region of the S1' pocket, which differs between bacterial PDFs and human PDF. Moreover, we analyzed the binding mode of 2 via NMR TITAN line shape analysis, revealing an induced fit mechanism.

Structure predictions and functional insights into Amidase_3 domain containing N-acetylmuramyl-L-alanine amidases from Deinococcus indicus DR1.

BACKGROUND: N-acetylmuramyl-L-alanine amidases are cell wall modifying enzymes that cleave the amide bond between the sugar residues and stem peptide in peptidoglycan. Amidases play a vital role in septal cell wall cleavage and help separate daughter cells during cell division. Most amidases are zinc metalloenzymes, and E. coli cells lacking amidases grow as chains with daughter cells attached to each other. In this study, we have characterized two amidase enzymes from Deinococcus indicus DR1. D. indicus DR1 is known for its high arsenic tolerance and unique cell envelope. However, details of their cell wall biogenesis remain largely unexplored. RESULTS: We have characterized two amidases Ami1Di and Ami2Di from D. indicus DR1. Both Ami1Di and Ami2Di suppress cell separation defects in E. coli amidase mutants, suggesting that these enzymes are able to cleave septal cell wall. Ami1Di and Ami2Di proteins possess the Amidase_3 catalytic domain with conserved -GHGG- motif and Zn2+ binding sites. Zn2+- binding in Ami1Di is crucial for amidase activity. AlphaFold2 structures of both Ami1Di and Ami2Di were predicted, and Ami1Di was a closer homolog to AmiA of E. coli. CONCLUSION: Our results indicate that Ami1Di and Ami2Di enzymes can cleave peptidoglycan, and structural prediction studies revealed insights into the activity and regulation of these enzymes in D. indicus DR1.

Engineering quorum quenching acylases with improved kinetic and biochemical properties.

Many Gram-negative bacteria use N-acyl-L-homoserine lactone (AHL) signals to coordinate phenotypes such as biofilm formation and virulence factor production. Quorum-quenching enzymes, such as AHL acylases, chemically degrade these molecules which prevents signal reception by bacteria and inhibits undesirable biofilm-related traits. These capabilities make acylases appealing candidates for controlling microbes, yet candidates with high activity levels and substrate specificity and that are capable of being formulated into materials are needed. In this work, we undertook engineering efforts against two AHL acylases, PvdQ and MacQ, to generate these improved properties using the Protein One-Stop Shop Server. The engineering of acylases is complicated by low-throughput enzymatic assays. Alleviating this challenge, we report a time-course kinetic assay for AHL acylases that monitors the real-time production of homoserine lactone. Using the assay, we identified variants of PvdQ that were significantly stabilized, with melting point increases of up to 13.2°C, which translated into high resistance against organic solvents and increased compatibility with material coatings. While the MacQ mutants were unexpectedly destabilized, they had considerably improved kinetic properties, with >10-fold increases against N-butyryl-L-homoserine lactone and N-hexanoyl-L-homoserine lactone. Accordingly, these changes resulted in increased quenching abilities using a biosensor model and greater inhibition of virulence factor production of Pseudomonas aeruginosa PA14. While the crystal structure of one of the MacQ variants, M1, did not reveal obvious structural determinants explaining the observed changes in kinetics, it allowed for the capture of an acyl-enzyme intermediate that confirms a previously hypothesized catalytic mechanism of AHL acylases.

Metabolism of toxic benzonitrile and hydroxybenzonitrile isomers via several distinct central pathway intermediates in a catabolically robust Burkholderia sp.

Aromatic nitriles are of considerable environmental concern, because of their hazardous impacts on the health of both humans and wildlife. In the present study, Burkholderia sp. strain BC1 was observed to be capable of utilizing toxic benzonitrile and hydroxybenzonitrile isomers singly, as sole carbon and energy sources. The results of chromatographic and spectrometric analyses in combination with oxygen uptake and enzyme activity studies, revealed the metabolism of benzonitrile as well as 2-, 3-, and 4-hydroxybenzonitriles by nitrile hydratase-amidase to the corresponding carboxylates. These carboxylates were further metabolized via central pathways, namely benzoate-catechol, salicylate-catechol, 3-hydroxybenzoate-gentisate and 4-hydroxybenzoate-protocatechute pathways in strain BC1, ultimately leading to the TCA cycle intermediates. Studies also evaluated substrate specificity profiles of both nitrile hydratase and amidase(s) involved in the denitrification of the nitriles. In addition, a few metabolic crosstalk events due to the induction of multiple operons by central metabolites were appraised in strain BC1. The present study illustrates the broad degradative potential of strain BC1, harboring diverse catabolic machinery of biotechnological importance, elucidating pathways for the assimilation of benzonitrile and that of hydroxybenzonitrile isomers for the first time.

Dual effect of anandamide on spinal nociceptive transmission in control and inflammatory conditions.

Anandamide (AEA) is an important modulator of nociception in the spinal dorsal horn, acting presynaptically through Cannabinoid (CB1) and Transient receptor potential vanilloid (TRPV1) receptors. The role of AEA (1 µM, 10 µM, and 30 µM) application on the modulation of nociceptive synaptic transmission under control and inflammatory conditions was studied by recording miniature excitatory postsynaptic currents (mEPSCs) from neurons in spinal cord slices. Inhibition of the CB1 receptors by PF514273, TRPV1 by SB366791, and the fatty acid amide hydrolase (FAAH) by URB597 was used. Under naïve conditions, the AEA application did not affect the mEPSCs frequency (1.43±0.12 Hz) when all the recorded neurons were considered. The mEPSC frequency increased (180.0±39.2%) only when AEA (30 µM) was applied with PF514273 and URB597. Analysis showed that one sub-population of neurons had synaptic input inhibited (39.1% of neurons), the second excited (43.5%), whereas 8.7% showed a mixed effect and 8.7% did not respond to the AEA. With inflammation, the AEA effect was highly inhibitory (72.7%), while the excitation was negligible (9.1%), and 18.2% were not modulated. After inflammation, more neurons (45.0%) responded even to low AEA by mEPSC frequency increase with PF514273/URB597 present. AEA-induced dual (excitatory/inhibitory) effects at the 1st nociceptive synapse should be considered when developing analgesics targeting the endocannabinoid system. These findings contrast the clear inhibitory effects of the AEA precursor 20:4-NAPE application described previously and suggest that modulation of endogenous AEA production may be more favorable for analgesic treatments.

Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

Signaling through the Salmonella PbgA-LapB regulatory complex activates LpxC proteolysis and limits lipopolysaccharide biogenesis during stationary-phase growth.

Salmonella enterica serovar Typhimurium (S. Typhimurium) controls lipopolysaccharide (LPS) biosynthesis by regulating proteolysis of LpxC, the rate-limiting enzyme and target of preclinical antibiotics. PbgA/YejM/LapC regulates LpxC levels and controls outer membrane (OM) LPS composition at the log-to-stationary phase transition. Suppressor substitutions in LPS assembly protein B (LapB/YciM) rescue the LPS and OM integrity defects of pbgA-mutant S. Typhimurium. We hypothesized that PbgA regulates LpxC proteolysis by controlling LapB's ability to bind LpxC as a function of the growth phase. According to existing models, when nutrients are abundant, PbgA binds and restricts LapB from interacting with LpxC and FtsH, which limits LpxC proteolysis. However, when nutrients are limited, there is debate whether LapB dissociates from PbgA to bind LpxC and FtsH to enhance degradation. We sought to examine these models and investigate how the structure of LapB enables salmonellae to control LpxC proteolysis and LPS biosynthesis. Salmonellae increase LapB levels during the stationary phase to promote LpxC degradation, which limits lipid A-core production and increases their survival. The deletion of lapB, resulting in unregulated lipid A-core production and LpxC overabundance, leads to bacterial growth retardation. Tetratricopeptide repeats near the cytosol-inner membrane interface are sufficient for LapB to bind LpxC, and remarkably, LapB and PbgA interact in both growth phases, yet LpxC only associates with LapB in the stationary phase. Our findings support that PbgA-LapB exists as a constitutive complex in S. Typhimurium, which differentially binds LpxC to control LpxC proteolysis and limit lipid A-core biosynthesis in response to changes in the environment.IMPORTANCEAntimicrobial resistance has been a costly setback for human health and agriculture. Continued pursuit of new antibiotics and targets is imperative, and an improved understanding of existing ones is necessary. LpxC is an essential target of preclinical trial antibiotics that can eliminate multidrug-resistant Gram-negative bacterial infections. LapB is a natural LpxC inhibitor that targets LpxC for degradation and limits lipopolysaccharide production in Enterobacteriaceae. Contrary to some studies, findings herein support that LapB remains in complex instead of dissociating from its presumed negative regulator, PbgA/YejM/LapC, under conditions where LpxC proteolysis is enhanced. Advanced comprehension of this critical protein-lipid signaling network will lead to future development and refinement of small molecules that can specifically interfere.

SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Genetic variation in endocannabinoid signaling: Anxiety, depression, and threat- and reward-related brain functioning during the transition into adolescence.

BACKGROUND: The endocannabinoid system modulates neural activity throughout the lifespan. In adults, neuroimaging studies link a common genetic variant in fatty acid amide hydrolase (FAAH C385A)-an enzyme that regulates endocannabinoid signaling-to reduced risk of anxiety and depression, and altered threat- and reward-related neural activity. However, limited research has investigated these associations during the transition into adolescence, a period of substantial neurodevelopment and increased psychopathology risk. METHODS: This study included FAAH genotype and longitudinal neuroimaging and neurobehavioral data from 4811 youth (46% female; 9-11 years at Baseline, 11-13 years at Year 2) from the Adolescent Brain Cognitive DevelopmentSM Study. Linear mixed models examined the effects of FAAH and the FAAH x time interaction on anxiety and depressive symptoms, amygdala reactivity to threatening faces, and nucleus accumbens (NAcc) response to happy faces during the emotional n-back task. RESULTS: A significant main effect of FAAH on depressive symptoms was observed, such that depressive symptoms were lower across both timepoints in those with the AA genotype compared to both AC and CC genotypes (p's<0.05). There were no significant FAAH x time interactions for anxiety, depression, or neural responses (p's>0.05). Additionally, there were no main effects of FAAH on anxiety or neural responses (p's>0.05). CONCLUSIONS: Our findings add to emerging evidence linking the FAAH C385A variant to lower risk of psychopathology, and extend these findings to a developmental sample. In particular, we found lower depressive symptoms in FAAH AA genotypes compared to AC and CC genotypes. Future research is needed to characterize the role of the FAAH variant and the eCB system more broadly in neurodevelopment and psychiatric risk.

Cloning and Characterization of Chitin Deacetylase from Euphausia superba.

Chitin deacetylase (CDA) can catalyze the deacetylation of chitin to produce chitosan. In this study, we identified and characterized a chitin deacetylase gene from Euphausia superba (EsCDA-9k), and a soluble recombinant protein chitin deacetylase from Euphausia superba of molecular weight 45 kDa was cloned, expressed, and purified. The full-length cDNA sequence of EsCDA-9k was 1068 bp long and encoded 355 amino acid residues that contained the typical domain structure of carbohydrate esterase family 4. The predicted three-dimensional structure of EsCDA-9k showed a 67.32% homology with Penaeus monodon. Recombinant chitin deacetylase had the highest activity at 40 °C and pH 8.0 in Tris-HCl buffer. The enzyme activity was enhanced by metal ions Co2+, Fe3+, Ca2+, and Na+, while it was inhibited by Zn2+, Ba2+, Mg2+, and EDTA. Molecular simulation of EsCDA-9k was conducted based on sequence alignment and homology modeling. The EsCDA-9k F18G mutant showed a 1.6-fold higher activity than the wild-type enzyme. In summary, this is the first report of the cloning and heterologous expression of the chitin deacetylase gene in Euphausia superba. The characterization and function study of EsCDA-9k will serve as an important reference point for future application.

Bile salt hydrolase acyltransferase activity expands bile acid diversity.

Bile acids (BAs) are steroid detergents in bile that contribute to the absorption of fats and fat-soluble vitamins while shaping the gut microbiome because of their antimicrobial properties1-4. Here we identify the enzyme responsible for a mechanism of BA metabolism by the gut microbiota involving amino acid conjugation to the acyl-site of BAs, thus producing a diverse suite of microbially conjugated bile acids (MCBAs). We show that this transformation is mediated by acyltransferase activity of bile salt hydrolase (bile salt hydrolase/transferase, BSH/T). Clostridium perfringens BSH/T rapidly performed acyl transfer when provided various amino acids and taurocholate, glycocholate or cholate, with an optimum at pH 5.3. Amino acid conjugation by C. perfringens BSH/T was diverse, including all proteinaceous amino acids except proline and aspartate. MCBA production was widespread among gut bacteria, with strain-specific amino acid use. Species with similar BSH/T amino acid sequences had similar conjugation profiles and several bsh/t alleles correlated with increased conjugation diversity. Tertiary structure mapping of BSH/T followed by mutagenesis experiments showed that active site structure affects amino acid selectivity. These MCBA products had antimicrobial properties, where greater amino acid hydrophobicity showed greater antimicrobial activity. Inhibitory concentrations of MCBAs reached those measured natively in the mammalian gut. MCBAs fed to mice entered enterohepatic circulation, in which liver and gallbladder concentrations varied depending on the conjugated amino acid. Quantifying MCBAs in human faecal samples showed that they reach concentrations equal to or greater than secondary and primary BAs and were reduced after bariatric surgery, thus supporting MCBAs as a significant component of the BA pool that can be altered by changes in gastrointestinal physiology. In conclusion, the inherent acyltransferase activity of BSH/T greatly diversifies BA chemistry, creating a set of previously underappreciated metabolites with the potential to affect the microbiome and human health.

DDAH1 recruits peroxiredoxin 1 and sulfiredoxin 1 to preserve its activity and regulate intracellular redox homeostasis.

Growing evidence suggests that dimethylarginine dimethylaminohydrolase 1 (DDAH1), a crucial enzyme for the degradation of asymmetric dimethylarginine (ADMA), is closely related to oxidative stress during the development of multiple diseases. However, the underlying mechanism by which DDAH1 regulates the intracellular redox state remains unclear. In the present study, DDAH1 was shown to interact with peroxiredoxin 1 (PRDX1) and sulfiredoxin 1 (SRXN1), and these interactions could be enhanced by oxidative stress. In HepG2 cells, H2O2-induced downregulation of DDAH1 and accumulation of ADMA were attenuated by overexpression of PRDX1 or SRXN1 but exacerbated by knockdown of PRDX1 or SRXN1. On the other hand, DDAH1 also maintained the expression of PRDX1 and SRXN1 in H2O2-treated cells. Furthermore, global knockout of Ddah1 (Ddah1-/-) or liver-specific knockout of Ddah1 (Ddah1HKO) exacerbated, while overexpression of DDAH1 alleviated liver dysfunction, hepatic oxidative stress and downregulation of PRDX1 and SRXN1 in CCl4-treated mice. Overexpression of liver PRDX1 improved liver function, attenuated hepatic oxidative stress and DDAH1 downregulation, and diminished the differences between wild type and Ddah1-/- mice after CCl4 treatment. Collectively, our results suggest that the regulatory effect of DDAH1 on cellular redox homeostasis under stress conditions is due, at least in part, to the interaction with PRDX1 and SRXN1.

Fatty Acid Amide Hydrolase and Cannabinoid Receptor Type 1 Genes Regulation is Modulated by Social Isolation in Rats.

Social isolation is a state of lack of social connections, involving the modulation of different molecular signalling cascades and associated with high risk of mental health issues. To investigate if and how gene expression is modulated by social experience at the central level, we analyzed the effects of 5 weeks of social isolation in rats focusing on endocannabinoid system genes transcription in key brain regions involved in emotional control. We observed selective reduction in mRNA levels for fatty acid amide hydrolase (Faah) and cannabinoid receptor type 1 (Cnr1) genes in the amygdala complex and of Cnr1 in the prefrontal cortex of socially isolated rats when compared to controls, and these changes appear to be partially driven by trimethylation of Lysine 27 and acetylation of Lysine 9 at Histone 3. The alterations of Cnr1 transcriptional regulation result also directly correlated with those of oxytocin receptor gene. We here suggest that to counteract the effects of SI, it is of relevance to restore the endocannabinoid system homeostasis via the use of environmental triggers able to revert those epigenetic mechanisms accounting for the alterations observed.

A fluorogenic substrate for the detection of lipid amidases in intact cells.

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 µM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 µM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 µM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Cryo-EM structures of Mycobacterium tuberculosis polynucleotide phosphorylase suggest a potential mechanism for its RNA substrate degradation.

As one of the oldest infectious diseases in the world, tuberculosis (TB) is the second most deadly infectious disease after COVID-19. Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), which can attack various organs of the human body. Up to now, drug-resistant TB continues to be a public health threat. Pyrazinamide (PZA) is regarded as a sterilizing drug in the treatment of TB due to its distinct ability to target Mtb persisters. Previously we demonstrated that a D67N mutation in Mycobacterium tuberculosis polynucleotide phosphorylase (MtbPNPase, Rv2783c) confers resistance to PZA and Rv2783c is a potential target for PZA, but the mechanism leading to PZA resistance remains unclear. To gain further insight into the MtbPNPase, we determined the cryo-EM structures of apo Rv2783c, its mutant form and its complex with RNA. Our studies revealed the Rv2783c structure at atomic resolution and identified its enzymatic functional groups essential for its phosphorylase activities. We also investigated the molecular mechanisms underlying the resistance to PZA conferred by the mutation. Our research findings provide structural and functional insights enabling the development of new anti-tuberculosis drugs.

Human induced pluripotent stem cell line (SDQLCHi064-A) derived from a patient with Canavan disease carrying c.556_559dup GTTC and c.919delA mutations in the ASPA gene.

Canavan disease (CD, OMIM# 271900) is an autosomal recessive neurodegenerative disorder caused by homozygous or compound heterozygous mutations in ASPA gene, which result in catalytic deficiency of the aspartoacylase enzyme and the accumulation of N-acetylaspartic acid (NAA). Clinical presentation varies according to the age of disease onset. Here, we generated a human induced pluripotent stem cell line (hiPSCs) SDQLCHi064-A from a 5-month old boy with CD carrying two novel frame shift mutations c.556_559dupGTTC (p.L187Rfs*5) and c.919delA (p.S307Vfs*24) of the ASPA gene, in order for us to better understanding the disease.

LpxC inhibition: Potential and opportunities with carbohydrate scaffolds.

Uridine diphosphate-3-O-(hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a key enzyme involved in the biosynthesis of lipid A, an essential building block, for the construction and assembly of the outer membrane (OM) of Gram-negative bacteria. The enzyme is highly conserved in almost all Gram-negative bacteria and hence has emerged as a promising target for drug discovery in the fight against multi-drug resistant Gram-negative infections. Since the first nanomolar LpxC inhibitor, L-161,240, an oxazoline-based hydroxamate, the two-decade-long ongoing search has provided valuable information regarding essential features necessary for inhibition. Although the design and structure optimization for arriving at the most efficacious inhibitor of this enzyme has made good use of different heterocyclic moieties, the use of carbohydrate scaffold is scant. This review briefly covers the advancement and progress made in LpxC inhibition. The field awaits the use of potential associated with carbohydrate-based scaffolds for LpxC inhibition and the discovery of anti-bacterial agents against Gram-negative infections.

NAAA-regulated lipid signaling in monocytes controls the induction of hyperalgesic priming in mice.

Circulating monocytes participate in pain chronification but the molecular events that cause their deployment are unclear. Using a mouse model of hyperalgesic priming (HP), we show that monocytes enable progression to pain chronicity through a mechanism that requires transient activation of the hydrolase, N-acylethanolamine acid amidase (NAAA), and the consequent suppression of NAAA-regulated lipid signaling at peroxisome proliferator-activated receptor-α (PPAR-α). Inhibiting NAAA in the 72 hours following administration of a priming stimulus prevented HP. This effect was phenocopied by NAAA deletion and depended on PPAR-α recruitment. Mice lacking NAAA in CD11b+ cells - monocytes, macrophages, and neutrophils - were resistant to HP induction. Conversely, mice overexpressing NAAA or lacking PPAR-α in the same cells were constitutively primed. Depletion of monocytes, but not resident macrophages, generated mice that were refractory to HP. The results identify NAAA-regulated signaling in monocytes as a control node in the induction of HP and, potentially, the transition to pain chronicity.