Trait-based study predicts glycerol/diol dehydratases as a key function of the gut microbiota of hindgut-fermenting carnivores.

BACKGROUND: Microbial pdu and cob-cbi-hem gene clusters encode the key enzyme glycerol/diol dehydratase (PduCDE), which mediates the transformation of dietary nutrients glycerol and 1,2-propanediol (1,2-PD) to a variety of metabolites, and enzymes for cobalamin synthesis, a co-factor and shared good of microbial communities. It was the aim of this study to relate pdu as a multipurpose functional trait to environmental conditions and microbial community composition. We collected fecal samples from wild animal species living in captivity with different gut physiology and diet (n = 55, in total 104 samples), determined occurrence and diversity of pdu and cob-cbi-hem using a novel approach combining metagenomics with quantification of metabolic and genetic biomarkers, and conducted in vitro fermentations to test for trait-based activity. RESULTS: Fecal levels of the glycerol transformation product 1,3-propanediol (1,3-PD) were higher in hindgut than foregut fermenters. Gene-based analyses indicated that pduC harboring taxa are common feature of captive wild animal fecal microbiota that occur more frequently and at higher abundance in hindgut fermenters. Phylogenetic analysis of genomes reconstructed from metagenomic sequences identified captive wild animal fecal microbiota as taxonomically rich with a total of 4150 species and > 1800 novel species but pointed at only 56 species that at least partially harbored pdu and cbi-cob-hem. While taxonomic diversity was highest in fecal samples of foregut-fermenting herbivores, higher pduC abundance and higher diversity of pdu/cbi-cob-hem related to higher potential for glycerol and 1,2-PD utilization of the less diverse microbiota of hindgut-fermenting carnivores in vitro. CONCLUSION: Our approach combining metabolite and gene biomarker analysis with metagenomics and phenotypic characterization identified Pdu as a common function of fecal microbiota of captive wild animals shared by few taxa and stratified the potential of fecal microbiota for glycerol/1,2-PD utilization and cobalamin synthesis depending on diet and physiology of the host. This trait-based study suggests that the ability to utilize glycerol/1,2-PD is a key function of hindgut-fermenting carnivores, which does not relate to overall community diversity but links to the potential for cobalamin formation. Video Abstract.

Differential salt stress resistance in male and female Salix linearistipularis plants: insights from transcriptome profiling and the identification of the 4-hydroxy-tetrahydrodipicolinate synthase gene.

MAIN CONCLUSION: Lysine plays an essential role in the growth differences between male and female S. linearistipularis plants under salt stress. Furthermore, SlDHDPS is identified as a vital gene contributing to the differences in saline-alkali tolerance between male and female plants of S. linearistipularis. Soil salinization is a significant problem that severely restricts agricultural production worldwide. High salinity and low nutrient concentrations consequently prevent the growth of most plant species. Salix linearistipularis is the only woody plant (shrub) naturally distributed in the saline-alkali lands of the Songnen Plain in Northeast China, and it is one of the few plants capable of thriving in soils with extremely high salt and alkaline pH (>9.0) levels. However, insufficient attention has been given to the interplay between salt and nitrogen in the growth and development of S. linearistipularis. Here, the male and female plants of S. linearistipularis were subjected to salt stress with nitrogen-starvation or nitrogen-supplement treatments, and it was found that nitrogen significantly affects the difference in salt tolerance between male and female plants, with nitrogen-starvation significantly enhancing the salt stress tolerance of female plants compared to male plants. Transcriptional analyses showed 66 differentially expressed nitrogen-responsive genes in female and male roots, with most of them showing sexual differences in expression patterns under salinity stress. RNA-seq and RT-qPCR analysis demonstrated that six genes had an opposite salt-induced expression pattern in female and male roots. The expression of the 4-hydroxy-tetrahydrodipicolinate synthase encoding gene (SlDHDPS) in female roots was higher than that in male roots. Further treatment with exogenous lysine could significantly alleviate the inhibitory effect of salt stress on the growth of female and male plants. These results indicate that the SlDHDPS in the nitrogen metabolism pathway is involved in the resistance of S. linearistipularis to salt stress, which lays a foundation for further exploring the mechanism of nitrogen on salt tolerance of S. linearistipularis, and has a significant reference value for saline-alkali land management and sustainable agricultural development.

Severe pulmonary arterial hypertension in congenital sideroblastic anemia from PUS1 mutation - a case report.

BACKGROUND: Myopathy, lactic acidosis and inherited sideroblastic anemia (MLASA) are a group of rare intriguing disorders with wider pathophysiological implications. One of the causes of MLASA is the mutation in PUS1 gene that encodes for pseudouridine synthase. This PUS1 mutation results in MLASA in which anemia and myopathy predominate. Severe pulmonary arterial hypertension has not been previously reported in patients with PUS1 gene mutation. CASE REPORT: A 17 year old girl with congenital sideroblastic anemia presented with worsening of breathlessness. Severe pulmonary artery hypertension was documented on investigations. A homozygous variant in exon 3 of gene PUS1,( chromosome 12:g.131932301 C > T c.430 C > T) was found on sanger sequencing. CONCLUSION: We document severe pulmonary arterial hypertension in a patient of congenital sideroblastic anemia from PUS1 gene. We hypothesis that cross talk with TGFb pathways might occur in PUS1 mutation, and that might cause severe PAH. This observation might have therapeutic implications.

Enhancing metabolic efficiency via novel constitutive promoters to produce protocatechuic acid in Escherichia coli.

The antioxidant molecule protocatechuic acid (PCA) can also serve as a precursor for polymer building blocks. PCA can be produced in Escherichia coli overexpressing 3-dehydroshikimate dehydratase (DSD), an enzyme that catalyses the transformation of 3-dehydroshikimate to PCA. Nevertheless, optimizing the expression rate of recombinant enzymes is a key factor in metabolic engineering when producing biobased chemicals. In this study, a degenerate synthetic promoter approach was investigated to improve further the production of PCA. By limited screening of a randomized promoter library made using pSEVA221 plasmid in E. coli, three novel synthetic constitutive promoters were selected that increased the PCA yield from glucose by 10-21% compared to the inducible T7-promoter. RT-qPCR analysis showed that the DSD gene, regulated by the synthetic promoters, had high expression during the exponential phase, albeit the gene expression level dropped 250-fold during stationary phase. Besides the increased product yield, the synthetic promoters avoided the need for a costly inducer for gene expression. Screening of the entire promoter library is likely to provide more positive hits. The study also shows that E. coli transformed with the DSD gene on either pSEVA221 or pCDFDuet plasmids exhibit background PCA levels (~ 0.04 g/L) in the absence of a transcriptional regulatory element. KEY POINTS: • Degenerate synthetic promoters are remarkable tools to produce protocatechuic acid. • The constitutive synthetic promoters did not affect the growth rate of the bacterial host. • The use of constitutive synthetic promoters avoids the need for the costly inducer.

Large-scale analysis of the PAC domain structure of arogenate dehydratases reveals their evolutionary patterns in angiosperms.

Arogenate dehydratase (ADT) is the key limiting enzyme of plant phenylalanine biosynthesis, but some ADTs display a prephenate decarboxylase/dehydratase activity-conferring (PAC) domain. The genome resources of 70 species were employed to identify genes and outline their characteristics, especially the number and type of PAC domain structures. We obtained 522 ADTs, and their size, exon number, amino acid number and putative protein isoelectric point greatly varied from 306 to 2520 bp, 1 to 15, 101 to 839 and 4.37 to 11.18, respectively. We classified the ADTs into Class α (without a PAC domain) (115, 22.0 %), ß (with a type I PAC domain) (244, 46.7 %) and γ (with a type II PAC domain) (163, 31.2 %), and their distribution frequencies exhibited large differences among various branches of angiosperms. We found that Class γ members are more conserved than Class ß members, although they commonly experienced multiple duplication events and strong purifying selection, which resulted in a small number, and the putative origin order was from Class α to ß and then to γ. In addition, the co-occurrence of both Class ß and γ members could ensure the survival of angiosperms, while their optimized composition and strategically intertwined regulation may facilitate core eudicot success.

Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily.

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid. We have shown earlier that the enzyme forms a dimer of dimers, in which the C-terminal histidine residue from one monomer participates in the formation of the active site of an adjacent monomer. The histidine appears to be conserved across the sequences of sugar acid dehydratases. To study the role of the C-terminus, five variants (H579A, H579F, H579L, H579Q, and H579W) were produced. All variants showed decreased activity for the tested sugar acid substrates, except the variant H579L on D-fuconate, which showed about 20% increase in activity. The reaction kinetic data showed that the substrate preference was slightly modified in H579L compared to the wild-type enzyme, demonstrating that the alternation of the substrate preference of sugar acid dehydratases is possible. In addition, a crystal structure of H579L was determined at 2.4 Å with a product analog 2-oxobutyrate. This is the first enzyme-ligand complex structure from an IlvD/EDD superfamily enzyme. The binding of 2-oxobutyrate suggests how the substrate would bind into the active site in the orientation, which could lead to the dehydration reaction. KEY POINTS: • Mutation of the last histidine at the C-terminus changed the catalytic activity of L-arabinonate dehydratase from R. leguminosarum bv. trifolii against various C5/C6 sugar acids. • The variant H579L of L-arabinonate dehydratase showed an alteration of substrate preferences compared with the wild type. • The first enzyme-ligand complex crystal structure of an IlvD/EDD superfamily enzyme was solved.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3.

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

H-NS involved in positive regulation of glycerol dehydratase gene expression in Klebsiella pneumoniae 2e.

Glycerol dehydratase is the key and rate-limiting enzyme in the 1,3-propanediol synthesis pathway of Klebsiella pneumoniae, which determined the producing rate and yield of 1,3-propanediol. However, the expression regulation mechanism of glycerol dehydratase gene dhaB remains poorly unknown. In this study, a histone-like nucleoid-structuring (H-NS) protein was identified and characterized as the positive transcription regulator for dhaB expression in K. pneumoniae 2e, which exhibited high tolerance against crude glycerol in our previous study. Deletion of hns gene significantly decreased the transcription level of dhaB in K. pneumoniae 2e, which led to a remarkable defect on strain growth, glycerol dehydratase activity, and 3-hydroxypropanal production during glycerol fermentation. The transcription level of dhaB was significantly up-regulated in crude glycerol relative to pure glycerol, while the inactivation of H-NS resulted in more negative effect for transcription level of dhaB in the former. Though the H-NS expression level was almost comparable in both substrates, its multimer state was reduced in crude glycerol relative to pure glycerol, suggesting that the oligomerization state of H-NS might have contributed for positive regulation of dhaB expression. Furthermore, electrophoretic mobility shift and DNase I footprinting assays showed that H-NS could directly bind to the upstream promoter region of dhaB by recognizing the AT-rich region. These findings provided new insight into the transcriptional regulation mechanism of H-NS for glycerol dehydratase expression in K. pneumoniae, which might offer new target for engineering bacteria to industrially produce 1,3-propanediol.IMPORTANCEThe biological production of 1,3-propanediol from glycerol by microbial fermentation shows great promising prospect on industrial application. Glycerol dehydratase catalyzes the penultimate step in glycerol metabolism and is regarded as one of the key and rate-limiting enzymes for 1,3-propanediol production. H-NS was reported as a pleiotropic modulator with negative effects on gene expression in most studies. Here, we reported for the first time that the expression of glycerol dehydratase gene is positively regulated by the H-NS. The results provide insight into a novel molecular mechanism of H-NS for positive regulation of glycerol dehydratase gene expression in K. pneumoniae, which holds promising potential for facilitating construction of engineering highly efficient 1,3-propanediol-producing strains.

Enhancing the stress resistance of nitrile hydratase from Rhodococcus ruber via SpyTag/SpyCatcher-mediated α- and ß- subunits ligation.

BACKGROUND: Nitrile Hydratase (NHase) is one of the most important industrial enzyme widely used in the petroleum exploitation field. The enzyme, composed of two unrelated α- and ß-subunits, catalyzes the conversion of acrylonitrile to acrylamide, releasing a significant amount of heat and generating the organic solvent product, acrylamide. Both the heat and acrylamide solvent have an impact on the structural stability of NHase and its catalytic activity. Therefore, enhancing the stress resistance of NHase to toxic substances is meaningful for the petroleum industry. METHODS AND RESULTS: To improve the thermo-stability and acrylamide tolerance of NHase, the two subunits were fused in vivo using SpyTag and SpyCatcher, which were attached to the termini of each subunit in various combinations. Analysis of the engineered strains showed that the C-terminus of ß-NHase is a better fusion site than the N-terminus, while the C-terminus of α-NHase is the most suitable site for fusion with a larger protein. Fusion of SpyTag and SpyCatcher to the C-terminus of ß-NHase and α-NHase, respectively, led to improved acrylamide tolerance and a slight enhancement in the thermo-stability of one of the engineered strains, NBSt. CONCLUSION: These results indicate that in vivo ligation of different subunits using SpyTag/SpyCatcher is a valuable strategy for enhancing subunit interaction and improving stress tolerance.

The fission yeast ortholog of Coilin, Mug174, forms Cajal body-like nuclear condensates and is essential for cellular quiescence.

The Cajal body, a nuclear condensate, is crucial for ribonucleoprotein assembly, including small nuclear RNPs (snRNPs). While Coilin has been identified as an integral component of Cajal bodies, its exact function remains unclear. Moreover, no Coilin ortholog has been found in unicellular organisms to date. This study unveils Mug174 (Meiosis-upregulated gene 174) as the Coilin ortholog in the fission yeast Schizosaccharomyces pombe. Mug174 forms phase-separated condensates in vitro and is often associated with the nucleolus and the cleavage body in vivo. The generation of Mug174 foci relies on the trimethylguanosine (TMG) synthase Tgs1. Moreover, Mug174 interacts with Tgs1 and U snRNAs. Deletion of the mug174+ gene in S. pombe causes diverse pleiotropic phenotypes, encompassing defects in vegetative growth, meiosis, pre-mRNA splicing, TMG capping of U snRNAs, and chromosome segregation. In addition, we identified weak homology between Mug174 and human Coilin. Notably, human Coilin expressed in fission yeast colocalizes with Mug174. Critically, Mug174 is indispensable for the maintenance of and transition from cellular quiescence. These findings highlight the Coilin ortholog in fission yeast and suggest that the Cajal body is implicated in cellular quiescence, thereby preventing human diseases.

Shy is a proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain.

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy copurified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When coexpressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to copurify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retro-aldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity toward C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted antiparallel ß-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo) heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

A new lysine biosynthetic enzyme from a bacterial endosymbiont shaped by genetic drift and genome reduction.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

Ectopic Production of 3,4-Dihydroxybenzoate in Planta Affects Cellulose Structure and Organization.

Lignocellulosic biomass is a highly sustainable and largely carbon dioxide neutral feedstock for the production of biofuels and advanced biomaterials. Although thermochemical pretreatment is typically used to increase the efficiency of cell wall deconstruction, genetic engineering of the major plant cell wall polymers, especially lignin, has shown promise as an alternative approach to reduce biomass recalcitrance. Poplar trees with reduced lignin content and altered composition were previously developed by overexpressing bacterial 3-dehydroshikimate dehydratase (QsuB) enzyme to divert carbon flux from the shikimate pathway. In this work, three transgenic poplar lines with increasing QsuB expression levels and different lignin contents were studied using small-angle neutron scattering (SANS) and wide-angle X-ray scattering (WAXS). SANS showed that although the cellulose microfibril cross-sectional dimension remained unchanged, the ordered organization of the microfibrils progressively decreased with increased QsuB expression. This was correlated with decreasing total lignin content in the QsuB lines. WAXS showed that the crystallite dimensions of cellulose microfibrils transverse to the growth direction were not affected by the QsuB expression, but the crystallite dimensions parallel to the growth direction were decreased by ∼20%. Cellulose crystallinity was also decreased with increased QsuB expression, which could be related to high levels of 3,4-dihydroxybenzoate, the product of QsuB expression, disrupting microfibril crystallization. In addition, the cellulose microfibril orientation angle showed a bimodal distribution at higher QsuB expression levels. Overall, this study provides new structural insights into the impact of ectopic synthesis of small-molecule metabolites on cellulose organization and structure that can be used for future efforts aimed at reducing biomass recalcitrance.

Expression of dehydroshikimate dehydratase in poplar induces transcriptional and metabolic changes in the phenylpropanoid pathway.

Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

Arogenate dehydratase isoforms strategically deregulate phenylalanine biosynthesis in Akebia trifoliata.

Arogenate dehydratase (ADT) is key for phenylalanine (Phe) biosynthesis in plants. To examine ADT components and function in Akebia trifoliata, a representative of Ranunculaceae, we first identified eight ADTs (AktADT1-8, encoding sequences varying from 1032 to 1962 bp) in the A. trifoliata reference genome and five proteins (AktADT1, AktADT4, AktADT7, AktADT8 and AktADT8s) with moonlighting prephenate dehydratase (PDT) activity and Km values varying from 0.43 to 2.17 mM. Structurally, two basic residue combinations (Val314/Ala317 and Ala314/Val317) in the PAC domain are essential for the moonlighting PDT activity of ADTs. Functionally, AktADT4 and AktADT8 successfully restored the wild-type phenotype of pha2, a knockout mutant of Saccharomyces cerevisiae. In addition, AktADTs are ubiquitously expressed, but their expression levels are tissue specific, and the half maximal inhibitory concentration (IC50) of Phe for AktADTs ranged from 49.81 to 331.17 µM. Both AktADT4 and AktADT8 and AktADT8s localized to chloroplast stromules and the cytosol, respectively, while the remaining AktADTs localized to the chloroplast stroma. These findings suggest that various strategies exist for regulating Phe biosynthesis in A. trifoliata. This provides a reasonable explanation for the high Phe content and insights for further genetic improvement of the edible fruits of A. trifoliata.

Structural and functional insights of itaconyl-CoA hydratase from Pseudomonas aeruginosa highlight a novel N-terminal hotdog fold.

Itaconyl-CoA hydratase in Pseudomonas aeruginosa (PaIch) converts itaconyl-CoA to (S)-citramalyl-CoA upon addition of a water molecule, a part of an itaconate catabolic pathway in virulent organisms required for their survival in humans host cells. Crystal structure analysis of PaIch showed that a unique N-terminal hotdog fold containing a 4-residue short helical segment α3-, named as an "eaten sausage", followed by a flexible loop region slipped away from the conserved ß-sheet scaffold, whereas the C-terminal hotdog fold is similar to all MaoC. A conserved hydratase motif with catalytic residues provides mechanistic insights into catalysis, and existence of a longer substrate binding tunnel may suggest the binding of longer CoA derivatives.

Mitochondrial tRNA pseudouridylation governs erythropoiesis.

ABSTRACT: Pseudouridine is the most prevalent RNA modification, and its aberrant function is implicated in various human diseases. However, the specific impact of pseudouridylation on hematopoiesis remains poorly understood. Here, we investigated the role of transfer RNA (tRNA) pseudouridylation in erythropoiesis and its association with mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA) pathogenesis. By using patient-specific induced pluripotent stem cells (iPSCs) carrying a genetic pseudouridine synthase 1 (PUS1) mutation and a corresponding mutant mouse model, we demonstrated impaired erythropoiesis in MLASA-iPSCs and anemia in the MLASA mouse model. Both MLASA-iPSCs and mouse erythroblasts exhibited compromised mitochondrial function and impaired protein synthesis. Mechanistically, we revealed that PUS1 deficiency resulted in reduced mitochondrial tRNA levels because of pseudouridylation loss, leading to aberrant mitochondrial translation. Screening of mitochondrial supplements aimed at enhancing respiration or heme synthesis showed limited effect in promoting erythroid differentiation. Interestingly, the mammalian target of rapamycin (mTOR) inhibitor rapamycin facilitated erythroid differentiation in MLASA-iPSCs by suppressing mTOR signaling and protein synthesis, and consistent results were observed in the MLASA mouse model. Importantly, rapamycin treatment partially ameliorated anemia phenotypes in a patient with MLASA. Our findings provide novel insights into the crucial role of mitochondrial tRNA pseudouridylation in governing erythropoiesis and present potential therapeutic strategies for patients with anemia facing challenges related to protein translation.

Structural and functional characterization of itaconyl-CoA hydratase and citramalyl-CoA lyase involved in itaconate metabolism of Pseudomonas aeruginosa.

Itaconate is a key anti-inflammatory/antibacterial metabolite in pathogen-macrophage interactions that induces adaptive changes in Pseudomonas aeruginosa-exposed airways. However, the impact and mechanisms underlying itaconate metabolism remain unclear. Our study reveals that itaconate significantly upregulates the expression of pyoverdine in P. aeruginosa and enhances its tolerance to tobramycin. Notably, the enzymes responsible for efficient itaconate metabolism, PaIch and PaCcl, play crucial roles in both utilizing itaconate and clearing its toxic metabolic intermediates. By using protein crystallography and molecular dynamics simulations analyses, we have elucidated the unique catalytic center and substrate-binding pocket of PaIch, which contribute to its highly efficient catalysis. Meanwhile, analysis of PaCcl has revealed how interactions between domains regulate the conformational changes of the active sites and binding pockets, influencing the catalytic process. Overall, our research uncovers the significance and mechanisms of PaIch and PaCcl in the efficient metabolism of itaconate by P. aeruginosa.

Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.