Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.

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.

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.
METHODS: 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.
CONCLUSIONS: 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.

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.

A VMYH motif was determined to help ketosynthases in polyketide assembly lines select α,β-unsaturated intermediates from an equilibrium mediated by an upstream dehydratase. Alterations of this motif decreased ketosynthase selectivity within a model tetraketide synthase, most significantly when replaced by the TNGQ motif of ketosynthases that accept D-β-hydroxy intermediates.

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 β-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.
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 β-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 β-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.
CONCLUSIONS: 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.

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.

The Krebs cycle enzyme aconitate decarboxylase 1 (ACOD1) mediates itaconate synthesis in monocytes and macrophages. Previously, we reported that administration of 4-octyl itaconate to lupus-prone mice abrogated immune dysregulation and clinical features. In this study, we explore the role of the endogenous ACOD1/itaconate pathway in the development of TLR7-induced lupus (imiquimod [IMQ] model). We found that, in vitro, ACOD1 was induced in mouse bone marrow-derived macrophages and human monocyte-derived macrophages following TLR7 stimulation. This induction was partially dependent on type I IFN receptor signaling and on specific intracellular pathways. In the IMQ-induced mouse model of lupus, ACOD1 knockout (Acod1-/-) displayed disruptions of the splenic architecture, increased serum levels of anti-dsDNA and proinflammatory cytokines, and enhanced kidney immune complex deposition and proteinuria, when compared with the IMQ-treated wild-type mice. Consistent with these results, Acod1-/- bone marrow-derived macrophages treated in vitro with IMQ showed higher proinflammatory features. Furthermore, itaconate serum levels in systemic lupus erythematosus patients were decreased compared with healthy individuals, in association with disease activity and specific perturbed cardiometabolic parameters. These findings suggest that the ACOD1/itaconate pathway plays important immunomodulatory and vasculoprotective roles in systemic lupus erythematosus, supporting the potential therapeutic role of itaconate analogs in autoimmune diseases.

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.

Compatible solutes are highly water-soluble organic osmolytes produced by microorganisms to adapt to extreme environments, such as high salinity and osmotic pressure. Among these, ectoine plays a crucial role in repairing and protecting nucleic acids, protein, biofilms, and cells. As a result, it has found widespread applications in cosmetics, biological agents, the enzyme industry, medicine, and other fields. Currently, the market value of ectoine is around US$ 1 000/kg, with a global demand reaching 15 000 tons per year. Although halophilic bacteria serve as the natural source of ectoine synthesis, its production in high-salinity media presents challenges such as equipment corrosion and high cost for industrial production. Advancements in functional genomics, systems biology, and synthetic biology have paved the way for the development of high-yielding cell factories through metabolic engineering, leading to significant progress. For example, engineered Escherichia coli achieved a maximum ectoine titer of 131.8 g/L, with a productivity of 1.37 g/(L·h). This review aims to explore the biosynthetic pathway, biochemical characteristics of key enzymes, and the biosynthesis of ectoine, shedding light on current research status and offering insights for industrial-scale ectoine production.