Substrate channeling enhances efficiency and prevents toxicity by directing unstable intermediates between active sites. Trimethylamine N-oxide demethylase (TDM) degrades trimethylamine N-oxide (TMAO) to dimethylamine and formaldehyde (HCHO), but the fate of HCHO has remained unclear. We report cryo-EM structures of TDM in apo, substrate-, and product-bound states that reveal a previously unknown channeling pathway. Combined structural, biochemical, and target molecular dynamics analyses show that HCHO is generated in a catalytic core and guided through a tunnel to a remote tetrahydrofolate (THF)-binding site, where it forms methylene-THF. Thus, TDM emerges as a bifunctional enzyme that unites TMAO demethylation with one-carbon transfer, providing a mechanistic explanation for its role in metabolic efficiency and detoxification.

Redox balance is crucial for normal development of stem and progenitor cells that reside in oxidative environments. This study explores the mechanisms of redox homeostasis in such niches and investigates myeloid-like blood progenitor cells that generate reactive oxygen species (ROS) and moderate it developmentally. Our findings reveal that during lymph gland development, as the blood-progenitor cells oxidize pyruvate via the TCA cycle leading to the generation of ROS, these cell also de novo synthesize GSH to counter excessive ROS and ensure redox balance. GABA metabolism, through GABA-shunt, restricts pyruvate dehydrogenase (PDH) activity and consequently TCA rate. This allows a metabolic route to sustain serine levels in these cells which is the rate limiting precursor controlling de novo GSH production. Disruption of GABA metabolism leads to a metabolic imbalance, characterized by excessive PDH activity, heightened TCA rate leading to impaired serine/GSH production and overall ROS dysregulation. Overall, the study presents a unique metabolic state whereby, in the blood-progenitor cells, by keeping PDH and TCA activity in check and promoting serine/GSH generation, GABA metabolism establishes a metabolic framework that optimises the use of ROS in blood-progenitors, while ensuring redox homeostasis. ### Competing Interest Statement The authors have declared no competing interest.

N -Acetylneuraminic acid (Neu5Ac) is a negatively charged nine-carbon amino sugar that is often the peripheral sugar in human cell-surface glycoconjugates. Some bacteria scavenge, import, and metabolize Neu5Ac or redeploy it on their cell surfaces for immune evasion. The import of Neu5Ac by many bacteria is mediated by tripartite ATP-independent periplasmic (TRAP) transporters. We have previously reported the structures of SiaQM, a membrane-embedded component of the Haemophilus influenzae TRAP transport system, (Currie et al., 2024). However, none of the published structures contain Neu5Ac bound to SiaQM. This information is critical for defining the transport mechanism and for further structure-activity relationship studies. Here, we report the structures of Fusobacterium nucleatum SiaQM with and without Neu5Ac. Both structures are in an inward (cytoplasmic side) facing conformation. The Neu5Ac-bound structure reveals the interactions of Neu5Ac with the transporter and its relationship with the Na + binding sites. Two of the Na + -binding sites are similar to those described previously. We identify a third metal-binding site that is further away and buried in the elevator domain. Ser300 and Ser345 interact with the C1-carboxylate group of Neu5Ac. Proteoliposome-based transport assays showed that Ser300-Neu5Ac interaction is critical for transport, whereas Ser345 is dispensable. Neu5Ac primarily interacts with residues in the elevator domain of the protein, thereby supporting the elevator with an operator mechanism. The residues interacting with Neu5Ac are conserved, providing fundamental information required to design inhibitors against this class of proteins.

Immune cells are increasingly recognized as nutrient sensors; however, their developmental role in regulating growth under homeostasis or dietary stress remains elusive. Here, we show that Drosophila larval macrophages, in response to excessive dietary sugar (HSD), reprogram their metabolic state by activating glycolysis, thereby enhancing TCA-cycle flux, and increasing lipogenesis-while concurrently maintaining a lipolytic state. Although this immune-metabolic configuration correlates with growth retardation under HSD, our genetic analyses reveal that enhanced lipogenesis supports growth, whereas glycolysis and lipolysis are growth-inhibitory. Notably, promoting immune-driven lipogenesis offsets early growth inhibition in imaginal discs caused by glycolytic and lipolytic immune-metabolic states. Our findings reveal a model of immune-metabolic imbalance, where growth-suppressive states (glycolysis, lipolysis) dominate over a growth-supportive lipogenic state, thereby impairing early organ size control and ultimately affecting adult size. Overall, this study provides important insights into dietary stress-induced immune-metabolic reprogramming and its link to organ size regulation and early developmental plasticity.

ATP-activated P2X3 receptors play a pivotal role in chronic cough, affecting more than 10% of the population. Despite the challenges posed by the highly conserved structure of P2X receptors, efforts to develop selective drugs targeting P2X3 have led to the development of camlipixant, a potent, selective P2X3 antagonist. However, the mechanisms of receptor desensitization, ion permeation, and structural basis of camlipixant binding to P2X3 remain unclear. Here, we report a cryo-EM structure of camlipixant-bound P2X3, revealing a previously undiscovered selective drug-binding site in the receptor. Our findings also demonstrate that conformational changes in the upper body domain, including the turret and camlipixantbinding pocket, play a critical role: turret opening facilitates P2X3 channel closure to a radius of 0.7 & Aring;, hindering cation transfer, whereas turret closure leads to channel opening. Structural and functional studies combined with molecular dynamics simulations provide a comprehensive understanding of camlipixant's selective inhibition of P2X3, offering a foundation for future drug development targeting this receptor.

Periplasmic nitrate reductase NapA is a member of the DMSO reductase (DMSOR) superfamily, which catalyzes the reduction of nitrate to nitrite. Campylobacter jejuni NapA (CjNapA) is notably larger compared to other structurally characterized NapA. Herein, we present the cryo-EM structure of CjNapA, the first of its kind from any ε-proteobacteria, revealing three lysine-rich insertions that could affect the substrate channel, potentially enhancing the affinity towards nitrate and other anionic substrates. Here, we report that wild-type CjNapA and NapA-C176D variants can reduce chlorate, perchlorate, and nitrate. However, the perchlorate and chlorate reductions by the CjNapA C176D variant are considerably slower, even though the perchlorate reductase has an Asp coordination to Mo. Molecular Dynamics (MD) simulations were performed to investigate the impact of the C176D mutation on substrate affinity and protein flexibility. Structural and kinetic comparisons with perchlorate reductase support evolutionary tuning for a desired function. Finally, structural comparisons with other structurally characterized NapAs also suggest the role of proximal pterin in CjNapA in electron transfer to the Mo center.

Redox balance is crucial for normal development of stem and progenitor cells that reside in oxidative environments. In this study, we explore the mechanisms of redox homeostasis in such niches and show that myeloid-like blood progenitor cells of the Drosophila larval lymph gland, that generate reactive oxygen species (ROS), moderate it developmentally by de novo synthesizing glutathione (GSH) to ensure redox balance. During lymph gland development, as the blood-progenitor cells oxidize pyruvate via the TCA cycle leading to the generation of ROS, GABA-shunt restricts pyruvate dehydrogenase (PDH) activity and consequently TCA cycle flux. This moderation enables a metabolic rerouting of TCA-derived oxaloacetate (OAA) to pyruvate via gluconeogenesis, which is necessary to sustain serine levels, the rate-limiting precursor for de novo GSH synthesis. Disruption of GABA metabolism causes metabolic imbalance, marked by excessive PDH activity and heightened TCA cycle flux. This results in reduced OAA availability, impaired gluconeogenic capacity, and insufficient serine/GSH production, ultimately leading to ROS dysregulation. Overall, this study identifies a unique metabolic framework in blood progenitor cells, where the GABA shunt, by restraining PDH and TCA cycle activity, maintains ROS at developmental levels. By coupling TCA-derived metabolites to GSH production, this state enables the TCA cycle to support both ROS generation and ROS scavenging, ensuring the developmental roles of ROS while preserving progenitor homeostasis.

Nutrient sensing and signaling play pivotal roles in animal growth. However, under dietary stress, this system falters, leading to growth defects. While immune cells are increasingly recognized as key nutrient sensors, their impact on animal growth remains poorly understood. In this study, we investigate how Drosophila larval macrophages respond to excessive dietary sugar and identify a reconfiguration of their metabolic state. They undergo a glycolytic shift, intensify TCA activity, and elevate TAG synthesis. While typical of sugarinduced nutrient stress, these changes interestingly exert contrasting effects on animal growth: glycolysis and increased TCA activity inhibit growth, while the lipogenic shift promotes it. However, the lipogenic response is insufficient to counteract the metabolic events suppressing growth, resulting in an overall reduction in adult fly size under high sugar conditions. Stimulating a pro-lipogenic immune state facilitates growth recovery, suggesting a growth paradigm governed by immune-metabolic transitions. This study unveils the unexpected influence of macrophage metabolic reprogramming on organismal growth homeostasis during Drosophila development, highlighting immune cell states as central determinants of growth, particularly under dietary stress.

Sialic acids play crucial roles in cell surface glycans of both eukaryotic and prokaryotic organisms, mediating various biological processes, including cell–cell interactions, development, immune response, oncogenesis and host–pathogen interactions. This review focuses on the β-anomeric form of N-acetylneuraminic acid (Neu5Ac), particularly its binding affinity towards various proteins, as elucidated by solved protein structures. Specifically, we delve into the binding mechanisms of Neu5Ac to proteins involved in sequestering and transporting Neu5Ac in Gram-negative bacteria, with implications for drug design targeting these proteins as antimicrobial agents. Unlike the initial assumptions, structural analyses revealed significant variability in the Neu5Ac binding pockets among proteins, indicating diverse evolutionary origins and binding modes. By comparing these findings with existing structures from other systems, we can effectively highlight the intricate relationship between protein structure and Neu5Ac recognition, emphasizing the need for tailored drug design strategies to inhibit Neu5Ac-binding proteins across bacterial species.

Rieske non-heme iron oxygenases are ubiquitously expressed in prokaryotes. These enzymes catalyze a wide variety of reactions, including cis-dihydroxylation, mono-hydroxylation, sulfoxidation, and demethylation. They contain a Rieske-type [2Fe-2S] cluster and an active site with a mono-nuclear iron bound to a 2-His carboxylate triad. Naphthalene 1,2 dioxygenase, a representative of this family, catalyzes the conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. This transformation requires naphthalene, two electrons, and an oxygen molecule. The first structure of the terminal oxygenase component of a Rieske non-heme iron oxygenase to be determined was naphthalene 1,2 dioxygenase (NDO-O). In this article, we describe in detail the methods used to recombinantly express and purify NDO-O in rich and minimal salts media, the crystallization of NDO-O for structure determination by X-ray crystallography, the challenges faced, and the methods used for the preparation of enzyme ligand complexes. The methods used here resulted in the determination of several NDO-O complexes with aromatic substrates, nitric oxide, oxygen molecule, and products, leading to an initial understanding of the mechanism of enzyme catalysis and the molecular determinants of the regio- and stereo-specificity of this class of enzymes.

Trimethylamine and its derivative trimethylamine N-oxide are widely present in marine and gut environments. These toxins, introduced from animal-derived food intake, have been linked to various health issues such fish odor syndrome, cardiovascular diseases, and gastrointestinal cancer. The microbiome has evolved into a metabolism that efficiently processes TMA as an energy and a carbon source, which has significant implications for bioenergy conversion and medical detoxification. However, the molecular mechanism of the process of turning over TMA in bacteria remains unclear due to the lack of structural insights. In this study, we obtained high-quality cryo-EM structures of trimethylamine N-oxide demethylase from Paracoccus sp. (paraTDM) in both apo- and substrate-bound conformations at 2.6 and 2.4 Å resolution, respectively. The paraTDM structure contains three distinct domains: the N-terminal, Core, and C-terminal domains. The N-terminal domain is involved in structural stability and assembly, while the C-terminal domain is a tetrahydrofolate-binding protein. The core domain binds to the substrate and to two metal ions, Zinc and Iron. The structures revealed that paraTDM adopts 2 + 21/2 complexation. The two half-domains of the complex contain only the C-terminal domain. Further biophysical and biochemical assays showed that paraTDM triggers breaking carbon-nitrogen bonds at the metal iron site. Overall, our study elucidated the molecular mechanism of the ubiquitous enzyme system involved in single-carbon transfer in prokaryotes and eukaryotes.