Plant metacaspases mediate programmed cell death in development, biotic and abiotic stresses, damage-induced immune response, and resistance to pathogen attack. Most metacaspases require Ca²⁺ for their activation and substrate processing. However, the Ca²⁺-dependent activation mechanism remains elusive. Here we report the crystal structures of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca²⁺-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca²⁺. Our analysis provides insight into the Ca²⁺-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses for engineering of more sustainable crops for food and biofuels.

Zinc is an essential micronutrient that supports all living organisms through regulating numerous biological processes. However, the mechanism of uptake regulation by intracellular Zn²⁺ status remains unclear. Here we report a cryo-electron microscopy structure of a ZIP-family transporter from Bordetella bronchiseptica at 3.05 Å resolution in an inward-facing, inhibited conformation. The transporter forms a homodimer, each protomer containing nine transmembrane helices and three metal ions. Two metal ions form a binuclear pore structure, and the third ion is located at an egress site facing the cytoplasm. The egress site is covered by a loop, and two histidine residues on the loop interact with the egress-site ion and regulate its release. Cell-based Zn²⁺ uptake and cell growth viability assays reveal a negative regulation of Zn²⁺ uptake through sensing intracellular Zn²⁺ status using a built-in sensor. These structural and biochemical analyses provide mechanistic insight into the autoregulation of zinc uptake across membranes.

Low catalytic activity is a key factor limiting the widespread application of type II L-asparaginase (ASNase) in the food and pharmaceutical industries. In this study, smart libraries were constructed by semi-rational design to improve the catalytic activity of type II ASNase from Bacillus licheniformis. Mutants with greatly enhanced catalytic efficiency were screened by saturation mutations and combinatorial mutations. A quintuple mutant ILRAC was ultimately obtained with specific activity of 841.62 IU/mg and k_cat/K_m of 537.15 min⁻¹·mM⁻¹, which were 4.24-fold and 6.32-fold more than those of wild-type ASNase. The highest specific activity and k_cat/K_m were firstly reported in type II ASNase from Bacillus licheniformis. Additionally, enhanced pH stability and superior thermostability were both achieved in mutant ILRAC. Meanwhile, structural alignment and molecular dynamic simulation demonstrated that high structure stability and strong substrate binding were beneficial for the improved thermal stability and enzymatic activity of mutant ILRAC. This is the first time that enzymatic activity of type II ASNase from Bacillus licheniformis has been enhanced by the semi-rational approach, and results provide new insights into enzymatic modification of L-asparaginase for industrial applications.