Biodegradation of ochratoxin A by Brevundimonas diminuta HAU429: Characterized performance, toxicity evaluation and functional enzymes.

Ochratoxin A (OTA) is a notorious mycotoxin commonly contaminating food products worldwide. In this study, an OTA-degrading strain Brevundimonas diminuta HAU429 was isolated by using hippuryl-L-phenylalanine as the sole carbon source. The biodegradation of OTA by strain HAU429 was a synergistic effect of intracellular and extracellular enzymes, which transformed OTA into ochratoxin α (OTα) through peptide bond cleavage. Cytotoxicity tests and cell metabolomics confirmed that the transformation of OTA into OTα resulted in the detoxification of its hepatotoxicity since OTA but not OTα disturbed redox homeostasis and induced oxidative damage to hepatocytes. Genome mining identified nine OTA hydrolase candidates in strain HAU429. They were heterologously expressed in Escherichia coli, and three novel amidohydrolase BT6, BT7 and BT9 were found to display OTA-hydrolyzing activity. BT6, BT7 and BT9 showed less than 45 % sequence identity with previously identified OTA-degrading amidohydrolases. BT6 and BT7 shared 60.9 % amino acid sequence identity, and exhibited much higher activity towards OTA than BT9. BT6 and BT7 could completely degrade 1 µg mL-1 of OTA within 1 h and 50 min, while BT9 hydrolyzed 100 % of OTA in the reaction mixture by 12 h. BT6 was the most thermostable retaining 38 % of activity after incubation at 70 °C for 10 min, while BT7 displayed the highest tolerance to ethanal remaining 76 % of activity in the presence of 6 % ethanol. This study could provide new insights towards microbial OTA degradation and promote the development of enzyme-catalyzed OTA detoxification during food processing.

Early and late-onset preeclampsia: effects of DDAH2 polymorphisms on ADMA levels and association with DDAH2 haplotypes.

Objective: To examine whether the DDAH2 promoter polymorphisms -1415G/A (rs2272592), -1151A/C (rs805304) and -449G/C (rs805305), and their haplotypes, are associated with PE compared with normotensive pregnant women, and whether they affect ADMA levels in these groups. Methods: A total of 208 pregnant women were included in the study and classified as early-onset (N=57) or late-onset PE (N =49), and as normotensive pregnant women (N = 102). Results: Pregnant with early-onset PE carrying the GC and GG genotypes for the DDAH2 -449G/C polymorphism had increased ADMA levels (P=0.01). No association of DDAH2 polymorphisms with PE in single-locus analysis was found. However, the G-C-G haplotype was associated with the risk for late-onset PE. Conclusion: It is suggested that DDAH2 polymorphisms could affect ADMA levels in PE, and that DDAH2 haplotypes may affect the risk for PE.

Cloning and functional characterization of the peptide deformylase encoding gene EuPDF1B from Eucommia ulmoides Oliv.

Peptide deformylase can catalyse the removal of formyl groups from the N-terminal formyl methionine of the primary polypeptide chain. The peptide deformylase genes of a few herbaceous plants have been studied to some extent, but the peptide deformylase genes of woody plants have not been studied. In this study, we isolated EuPDF1B from Eucommia ulmoides Oliv. The full-length sequence of EuPDF1B is 1176 bp long with a poly-A tail and contains an open reading frame of 831 bp that encodes a protein of 276 amino acids. EuPDF1B was localized to the chloroplast. qRT‒PCR analysis revealed that this gene was expressed in almost all tissues tested but mainly in mature leaves. Moreover, the expression of EuPDF1B was enhanced by ABA, MeJA and GA and inhibited by shading treatment. The expression pattern of EuPDF1B was further confirmed in EuPDF1Bp: GUS transgenic tobacco plants. Among all the transgenic tobacco plants, EuPDF1Bp-3 showed the highest GUS histochemical staining and activity in different tissues. This difference may be related to the presence of enhancer elements in the region from - 891 bp to - 236 bp of the EuPDF1B promoter. In addition, the expression of the chloroplast gene psbA and the net photosynthetic rate, fresh weight and height of tobacco plants overexpressing EuPDF1B were greater than those of the wild-type tobacco plants, suggesting that EuPDF1B may promote the growth of transgenic tobacco plants. This is the first time that PDF and its promoter have been cloned from woody plants, laying a foundation for further analysis of the function of PDF and the regulation of its expression.

A novel N-acetylglucosamine-6-phosphate deacetylase that is essential for chitin utilization in the chitinolytic bacterium, Chitiniphilus shinanonensis.

AIMS: We aimed to investigate the function of an unidentified gene annotated as a PIG-L domain deacetylase (cspld) in Chitiniphilus shinanonensis SAY3. cspld was identified using transposon mutagenesis, followed by negatively selecting a mutant incapable of growing on chitin, a polysaccharide consisting of N-acetyl-d-glucosamine (GlcNAc). We focused on the physiological role of CsPLD protein in chitin utilization. METHODS AND RESULTS: Recombinant CsPLD expressed in Escherichia coli exhibited GlcNAc-6-phosphate deacetylase (GPD) activity, which is involved in the metabolism of amino sugars. However, SAY3 possesses two genes (csnagA1 and csnagA2) in its genome that code for proteins whose primary sequences are homologous to those of typical GPDs. Recombinant CsNagA1 and CsNagA2 also exhibited GPD activity with 23 and 1.6% of catalytic efficiency (kcat/Km), respectively, compared to CsPLD. The gene-disrupted mutant, Δcspld was unable to grow on chitin or GlcNAc, whereas the three mutants, ΔcsnagA1, ΔcsnagA2, and ΔcsnagA1ΔcsnagA2 grew similarly to SAY3. The determination of GPD activity in the crude extracts of each mutant revealed that CsPLD is a major enzyme that accounts for almost all cellular activities. CONCLUSIONS: Deacetylation of GlcNAc-6P catalyzed by CsPLD (but not by typical GPDs) is essential for the assimilation of chitin and its constituent monosaccharide, GlcNAc, as a carbon and energy source in C. shinanonensis.

Transcriptomic signature, bioactivity and safety of a non-hepatotoxic analgesic generating AM404 in the midbrain PAG region.

Safe and effective pain management is a critical healthcare and societal need. The potential for acute liver injury from paracetamol (ApAP) overdose; nephrotoxicity and gastrointestinal damage from chronic non-steroidal anti-inflammatory drug (NSAID) use; and opioids' addiction are unresolved challenges. We developed SRP-001, a non-opioid and non-hepatotoxic small molecule that, unlike ApAP, does not produce the hepatotoxic metabolite N-acetyl-p-benzoquinone-imine (NAPQI) and preserves hepatic tight junction integrity at high doses. CD-1 mice exposed to SRP-001 showed no mortality, unlike a 70% mortality observed with increasing equimolar doses of ApAP within 72 h. SRP-001 and ApAP have comparable antinociceptive effects, including the complete Freund's adjuvant-induced inflammatory von Frey model. Both induce analgesia via N-arachidonoylphenolamine (AM404) formation in the midbrain periaqueductal grey (PAG) nociception region, with SRP-001 generating higher amounts of AM404 than ApAP. Single-cell transcriptomics of PAG uncovered that SRP-001 and ApAP also share modulation of pain-related gene expression and cell signaling pathways/networks, including endocannabinoid signaling, genes pertaining to mechanical nociception, and fatty acid amide hydrolase (FAAH). Both regulate the expression of key genes encoding FAAH, 2-arachidonoylglycerol (2-AG), cannabinoid receptor 1 (CNR1), CNR2, transient receptor potential vanilloid type 4 (TRPV4), and voltage-gated Ca2+ channel. Phase 1 trial (NCT05484414) (02/08/2022) demonstrates SRP-001's safety, tolerability, and favorable pharmacokinetics, including a half-life from 4.9 to 9.8 h. Given its non-hepatotoxicity and clinically validated analgesic mechanisms, SRP-001 offers a promising alternative to ApAP, NSAIDs, and opioids for safer pain treatment.

Probing the enzymatic activity and maturation process of the EcAIII Ntn-amidohydrolase using local random mutagenesis.

This report describes a comprehensive approach to local random mutagenesis of the E. coli Ntn-amidohydrolase EcAIII, and supplements the results published earlier for the randomization series RDM1. Here, random mutagenesis was applied in the center of the EcAIII molecule, i.e., in the region important for substrate binding and its immediate neighborhood (series RDM2, RDM3, RDM7), in the vicinity of the catalytic threonine triplet (series RDM4, RDM5, RDM6), in the linker region (series RDM8), and in the sodium-binding (stabilization) loop (series RDM9). The results revealed that the majority of the new EcAIII variants have abolished or significantly reduced rate of autoprocessing, even if the mutation was not in a highly conserved sequence and structure regions. AlphaFold-predicted structures of the mutants suggest the role of selected residues in the positioning of the linker and stabilization of the scissile bond in precisely correct orientation, enabling the nucleophilic attack during the maturation process. The presented data highlight the details of EcAIII geometry that are important for the autoproteolytic maturation and for the catalytic mechanism in general, and can be treated as a guide for protein engineering experiments with other Ntn-hydrolases.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

Targeting the GPI transamidase subunit GPAA1 abrogates the CD24 immune checkpoint in ovarian cancer.

CD24 is frequently overexpressed in ovarian cancer and promotes immune evasion by interacting with its receptor Siglec10, present on tumor-associated macrophages, providing a "don't eat me" signal that prevents targeting and phagocytosis by macrophages. Factors promoting CD24 expression could represent novel immunotherapeutic targets for ovarian cancer. Here, using a genome-wide CRISPR knockout screen, we identify GPAA1 (glycosylphosphatidylinositol anchor attachment 1), a factor that catalyzes the attachment of a glycosylphosphatidylinositol (GPI) lipid anchor to substrate proteins, as a positive regulator of CD24 cell surface expression. Genetic ablation of GPAA1 abolishes CD24 cell surface expression, enhances macrophage-mediated phagocytosis, and inhibits ovarian tumor growth in mice. GPAA1 shares structural similarities with aminopeptidases. Consequently, we show that bestatin, a clinically advanced aminopeptidase inhibitor, binds to GPAA1 and blocks GPI attachment, resulting in reduced CD24 cell surface expression, increased macrophage-mediated phagocytosis, and suppressed growth of ovarian tumors. Our study highlights the potential of targeting GPAA1 as an immunotherapeutic approach for CD24+ ovarian cancers.

Hydrophobic substrate binding pocket remodeling of echinocandin B deacylase based on multi-dimensional rational design.

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates.

Asp-tRNAAsn/Glu-tRNAGln amidotransferase A subunit-like amidase mediates the degradation of insecticide flonicamid by Variovorax boronicumulans CGMCC 4969.

The main metabolic product of the pyridinecarboxamide insecticide flonicamid, N-(4-trifluoromethylnicotinyl)glycinamide (TFNG-AM), has been shown to have very high mobility in soil, leading to its accumulation in the environment. Catabolic pathways of flonicamid have been widely reported, but few studies have focused on the metabolism of TFNG-AM. Here, the rapid transformation of TFNG-AM and production of the corresponding acid product N-(4-trifluoromethylnicotinoyl) glycine (TFNG) by the plant growth-promoting bacterium Variovorax boronicumulans CGMCC 4969 were investigated. With TFNG-AM at an initial concentration of 0.86 mmol/L, 90.70 % was transformed by V. boronicumulans CGMCC 4969 resting cells within 20 d, with a degradation half-life of 4.82 d. A novel amidase that potentially mediated this transformation process, called AmiD, was identified by bioinformatic analyses. The gene encoding amiD was cloned and expressed recombinantly in Escherichia coli, and the enzyme AmiD was characterized. Key amino acid residue Val154, which is associated with the catalytic activity and substrate specificity of signature family amidases, was identified for the first time by homology modeling, structural alignment, and site-directed mutagenesis analyses. When compared to wild-type recombinant AmiD, the mutant AmiD V154G demonstrated a 3.08-fold increase in activity toward TFNG-AM. The activity of AmiD V154G was greatly increased toward aromatic L-phenylalanine amides, heterocyclic TFNG-AM and IAM, and aliphatic asparagine, whereas it was dramatically lowered toward benzamide, phenylacetamide, nicotinamide, acetamide, acrylamide, and hexanamid. Quantitative PCR analysis revealed that AmiD may be a substrate-inducible enzyme in V. boronicumulans CGMCC 4969. The mechanism of transcriptional regulation of AmiD by a member of the AraC family of regulators encoded upstream of the amiD gene was preliminarily investigated. This study deepens our understanding of the mechanisms of metabolism of toxic amides in the environment, providing new ideas for microbial bioremediation.

Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

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.

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.

Dissection of N-deacetylase and N-sulfotransferase activities of NDST1 and their effects on Wnt8 distribution and signaling in Xenopus embryos.

Wnt is a family of secreted signaling proteins involved in the regulation of cellular processes, including maintenance of stem cells, carcinogenesis, and cell differentiation. In the context of early vertebrate embryogenesis, graded distribution of Wnt proteins has been thought to regulate positional information along the antero-posterior axis. However, understanding of the molecular basis for Wnt spatial distribution remains poor. Modified states of heparan sulfate (HS) proteoglycans are essential for Wnt8 localization, because depletion of N-deacetylase/N-sulfotransferase 1 (NDST1), a modification enzyme of HS chains, decreases Wnt8 levels and NDST1 overexpression increases Wnt8 levels on the cell surface. Since overexpression of NDST1 increases both deacetylation and N-sulfation of HS chains, it is not clear which function of NDST1 is actually involved in Wnt8 localization. In the present study, we generated an NDST1 mutant that specifically increases deacetylation, but not N-sulfation, of HS chains in Xenopus embryos. Unlike wild-type NDST1, this mutant did not increase Wnt8 accumulation on the cell surface, but it reduced canonical Wnt signaling, as determined with the TOP-Flash reporter assay. These results suggest that N-sulfation of HS chains is responsible for localization of Wnt8 and Wnt8 signaling, whereas deacetylation has an inhibitory effect on canonical Wnt signaling. Consistently, overexpression of wild-type NDST1, but not the mutant, resulted in small eyes in Xenopus embryos. Thus, our NDST1 mutant enables us to dissect the regulation of Wnt8 localization and signaling by HS proteoglycans by specifically manipulating the enzymatic activities of NDST1.

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.

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.

Engineering of a chitin deacetylase to generate tailor-made chitosan polymers.

Chitin deacetylases (CDAs) emerge as a valuable tool to produce chitosans with a nonrandom distribution of N-acetylglucosamine (GlcNAc) and glucosamine (GlcN) units. We hypothesized before that CDAs tend to bind certain sequences within the substrate matching their subsite preferences for either GlcNAc or GlcN units. Thus, they deacetylate or N-acetylate their substrates at nonrandom positions. To understand the molecular basis of these preferences, we analyzed the binding site of a CDA from Pestalotiopsis sp. (PesCDA) using a detailed activity screening of a site-saturation mutagenesis library. In addition, molecular dynamics simulations were conducted to get an in-depth view of crucial interactions along the binding site. Besides elucidating the function of several amino acids, we were able to show that only 3 residues are responsible for the highly specific binding of PesCDA to oligomeric substrates. The preference to bind a GlcNAc unit at subsite -2 and -1 can mainly be attributed to N75 and H199, respectively. Whereas an exchange of N75 at subsite -2 eliminates enzyme activity, H199 can be substituted with tyrosine to increase the GlcN acceptance at subsite -1. This change in substrate preference not only increases enzyme activity on certain substrates and changes composition of oligomeric products but also significantly changes the pattern of acetylation (PA) when N-acetylating polyglucosamine. Consequently, we could clearly show how subsite preferences influence the PA of chitosans produced with CDAs.

Insights from the molecular mechanism of pyrazinamide to mutated pyrazinamidase linked to the pncA gene in clinical isolates of Mycobacterium tuberculosis.

This study aims to conduct a comprehensive molecular dynamics strategy to evaluate whether mutations found in pyrazinamide monoresistant (PZAMR) strains of Mycobacterium tuberculosis (MTB) can potentially reduce the effectiveness of pyrazinamide (PZA) for tuberculosis (TB) treatment. Five single point mutations of pyrazinamidase (PZAse), an enzyme which is responsible for the activation of prodrug PZA into pyrazinoic acid, found in MTB clinical isolates, namely His82Arg, Thr87Met, Ser66Pro, Ala171Val, and Pro62Leu, were analyzed by the dynamics simulations both in the apo state (unbound state) and in the PZA bound state. The results showed that the mutation of His82 to Arg, Thr87 to Met, and Ser66 to Pro in PZAse affects the coordination state of the Fe2+ ion, which is a cofactor required for enzyme activity. These mutations change the flexibility, stability, and fluctuation of His51, His57, and ASP49 amino acid residues around the Fe2+ ion, culminating in an unstable complex and dissociation of PZA from the PZAse binding site. However, mutations of Ala171 to Val and Pro62 to Leu were found to have no effect on the complex's stability. Based on the results, PZAse mutations of His82Arg, Thr87Met, and Ser66Pro culminated in weak binding affinity for PZA and caused significant structural deformations that led to PZA resistance. Future structural and functional studies, as well as investigations into other aspects of drug resistance in PZAse, will require experimental clarification.Communicated by Ramaswamy H. Sarma.

Emerging insight of whole genome sequencing coupled with protein structure prediction into the pyrazinamide-resistance signature of Mycobacterium tuberculosis.

Pyrazinamide (PZA) is considered to be a pivotal drug to shorten the treatment of both drug-susceptible and drug-resistant tuberculosis, but its use is challenged by the reliability of drug-susceptibility testing (DST). PZA resistance in Mycobacterium tuberculosis (MTB) is relevant to the amino acid substitution of pyrazinamidase that is responsible for the conversion of PZA to active pyrazinoic acid (POA). The single nucleotide variants (SNVs) within ribosomal protein S1 (rpsA) or aspartate decarboxylase (panD), the binding targets of POA, has been reported to drive the PZA-resistance signature of MTB. In this study, whole genome sequencing (WGS) was used to identify SNVs within the pncA, rpsA and panD genes in 100 clinical MTB isolates associated with DST results for PZA. The potential influence of high-confidence, interim-confidence or emerging variants on the interplay between target genes and PZA or POA was simulated computationally, and predicted with a protein structure modelling approach. The DST results showed weak agreement with the identification of high-confidence variants within the pncA gene (Cohen's kappa coefficient=0.58), the analytic results of WGS coupled with protein structure modelling on pncA mutants (Cohen's kappa coefficient=0.524) or related genes (Cohen's kappa coefficient=0.504). Taken together, these results suggest the practicable application of a genotypic-coupled bioinformatic approach to manage PZA-containing regimens for patients with MTB.

NAAG synthetase deficiency has only low influence on pathogenesis in a Canavan disease mouse model.

Canavan disease (CD) is a leukodystrophy caused by mutations in the N-acetylaspartate (NAA) hydrolase aspartoacylase (ASPA). Inability to degrade NAA and its accumulation in the brain results in spongiform myelin degeneration. NAA is mainly synthesized by neurons, where it is also a precursor of the neuropeptide N-acetylaspartylglutamate (NAAG). Hydrolysis of this peptide by glutamate carboxypeptidases is an additional source of extracellular NAA besides the instant neuronal release of NAA. This study examines to what extent NAA released from NAAG contributes to NAA accumulation and pathogenesis in the brain of Aspanur7/nur7 mutant mice, an established model of CD. Towards this aim, Aspanur7/nur7 mice with additional deficiencies in NAAG synthetase genes Rimklb and/or Rimkla were generated. Loss of myelin in Aspanur7/nur7 mice was not significantly affected by Rimkla and Rimklb deficiency and there was also no obvious change in the extent of brain vacuolation. Astrogliosis was slightly reduced in the forebrain of Rimkla and Rimklb double deficient Aspanur7/nur7 mice. However, only minor improvements at the behavioral level were found. The brain NAA accumulation in CD mice was, however, not significantly reduced in the absence of NAAG synthesis. In summary, there was only a weak tendency towards reduced pathogenic symptoms in Aspanur7/nur7 mice deficient in NAAG synthesis. Therefore, we conclude that NAAG metabolism has little influence on NAA accumulation in Aspanur7/nur7 mice and development of pathological symptoms in CD.