Cells organize metabolism within a crowded intracellular milieu, yet the structural logic and functional consequences of supramolecular enzyme assemblies in vivo remain poorly defined. Here, using in situ cryo-electron tomography, we identify a previously uncharacterized high-order architecture of urate oxidase in mouse liver. Urate oxidase homotetramers assemble into helical fibers that tile laterally to form a stable, porous lamellar scaffold. This organization preserves extensive accessible surface area while permitting efficient substrate and product exchange. Structure-guided perturbation abolishes the assembly in vivo without measurably altering intrinsic catalytic activity, indicating a primary role in spatial organization rather than allosteric regulation. Consistent with this, the assembled state enhances resistance to thermal, proteolytic and oxidative stress, supporting sustained activity in the peroxide-rich peroxisomal matrix. Conserved across mammals, this architecture reveals a strategy for metabolic compartmentalization that couples efficient catalysis to increased molecular robustness without the trade-off between supramolecular assembly and enzyme accessibility. Enzyme organization inside cells remains poorly understood. Here, the authors use cryo-electron tomography to show that urate oxidase forms conserved helix-tiling assemblies in liver peroxisomes, stabilizing the enzyme while preserving efficient catalysis.
Chlorophyll synthase (ChlG) is a transmembrane enzyme that attaches the tetraprenyl tail to chlorophyllide to produce chlorophyll a, the key pigment of oxygenic photosynthesis. In the cyanobacterium Synechocystis sp. PCC 6803, ChlG forms a complex with the light-harvesting complex (LHC)-like high-light-inducible protein (Hlip) HliD. Here, to understand the catalytic mechanism of ChlG and the role of HliD, we used cryogenic electron microscopy to determine the structure of a ChlG2HliD2 complex in substrate-free and geranylgeranyl-pyrophosphate-bound states and AlphaFold to model binding of chlorophyllide at the active site of ChlG. In combination with site-directed mutagenesis and molecular dynamics simulations, our structural data allow us to propose the mechanisms of substrate specificity and catalysis by ChlG. Our structures also reveal how a central HliD homodimer binds two ChlG monomers, as well as the arrangement of the four chlorophyll and two carotenoid pigments bound by the Hlips, and shed light on the evolution of eukaryotic LHC antennae. The molecular mechanisms underlying chlorophyll a biosynthesis, photoprotection and transfer to photosystems are unclear. Now, cryogenic electron microscopy structures of cyanobacterial chlorophyll synthase bound to the light-harvesting complex (LHC)-like high-light-inducible protein HliD, chlorophyll and carotenoid molecules shed light on these mechanisms and the evolution of eukaryotic LHCs.
Chlorophyll is essential for photosynthesis but hazardous when unbound. A cryo-electron microscopy structure of chlorophyll synthase with the photoprotective HliD protein reveals a molecular link between pigment synthesis, safe chlorophyll handling and the evolution of light-harvesting antennae.
The cell membrane governs biochemical processes and is instrumental for regulating immune cell functions. Its spatial organization is determined by lipid composition and membrane fluidity. A new fluorescence-based single-cell method links membrane fluidity to cellular function and adds a new dimension to immunological studies.
Peroxisomes are highly heterogeneous organelles with remarkable functional diversity across species and tissues. Isolating peroxisomes is essential for proteomic investigating their biological roles and regulatory mechanisms. Here, we discover that the N-terminal region of ATF6α interacts directly with peroxisomal membrane protein ABCD1 and ABCD3 in vitro and develop an affinity-based strategy PerCap, leveraging these interactions to rapidly purify native peroxisomes. Using this approach, we isolated peroxisomes from human cells, validated their intactness and identified the potential peroxisomal proteins by proteomic analysis. We show that AKAP1, CPT1A and CYB5R1 also localize to peroxisomes and loss of AKAP1 affects peroxisome morphology. Peroxisomes were also isolated from mouse tissues to compare tissue-specific peroxisomal proteins. By monitoring peroxisomal proteome dynamics during Torin1-induced pexophagy, we identified UBE2V1 as a contributor to this process. In summary, PerCap enables isolation of native peroxisomes from human cells and mouse tissues for proteomic and functional analyses. Peroxisomes are organelles with remarkable functional diversity across species and tissues. A peroxisome capture method to enrich native peroxisomes from human cells and mouse tissues enables functional and proteomic analysis.
