Highlights d Genetics, biochemistry, proteomics, and cryo-EM define GID E3 ligase regulation d Carbon stress induces assembly of an inactive anticipatory GID Ant complex d Environmental perturbations trigger substrate receptor assembly into active GID E3s d Structural model of N-degron substrate ubiquitylation by multisubunit RING-RING E3

Most bacteria contain a peptidoglycan (PG) cell wall, which is critical for maintenance of shape and important for cell division. In contrast, Planctomycetes have been proposed to produce a proteinaceous cell wall devoid of PG. The apparent absence of PG has been used as an argument for the putative planctomycetal ancestry of all bacterial lineages. Here we show, employing multiple bioinformatic methods, that planctomycetal genomes encode proteins required for PG synthesis. Furthermore, we biochemically demonstrate the presence of the sugar and the peptide components of PG in Planctomycetes. In addition, light and electron microscopic experiments reveal planctomycetal PG sacculi that are susceptible to lysozyme treatment. Finally, cryo-electron tomography demonstrates that Planctomycetes possess a typical PG cell wall and that their cellular architecture is thus more similar to that of other Gram-negative bacteria. Our findings shed new light on the cellular architecture and cell division of the maverick Planctomycetes.

The capture of CO2 by carboxylases is key to sustainable biocatalysis and a carbon-neutral bio-economy, yet currently limited to few naturally existing enzymes. Here, we developed glycolyl-CoA carboxylase (GCC), a new-to-nature enzyme, by combining rational design, high-throughput microfluidics and microplate screens. During this process, GCC’s catalytic efficiency improved by three orders of magnitude to match the properties of natural CO2-fixing enzymes. We verified our active-site redesign with an atomic-resolution, 1.96-Å cryo-electron microscopy structure and engineered two more enzymes that, together with GCC, form a carboxylation module for the conversion of glycolate (C2) to glycerate (C3). We demonstrate how this module can be interfaced with natural photorespiration, ethylene glycol conversion and synthetic CO2 fixation. Based on stoichiometrical calculations, GCC is predicted to increase the carbon efficiency of all of these processes by up to 150% while reducing their theoretical energy demand, showcasing how expanding the solution space of natural metabolism provides new opportunities for biotechnology and agriculture.

Qiao et al. (Schulman) -Interconversion between anticipatory and active GID E3 ubiquitin ligase conformations via metabolically-driven substrate receptor assembly 2 SUMMARY Cells respond to environmental changes by toggling metabolic pathways, preparing for homeostasis, and anticipating future stresses. For example, in Saccharomyces cerevisiae, carbon stress-induced gluconeogenesis is terminated upon glucose availability, a process that involves the multiprotein E3 ligase, GID SR4 , recruiting N-termini and catalyzing ubiquitylation of gluconeogenic enzymes. Here, genetics, biochemistry, and cryo electron microscopy define molecular underpinnings of glucose-induced degradation. Unexpectedly, carbon stress induces an inactive anticipatory complex (GID Ant ), which awaits a glucoseinduced substrate receptor to form the active GID SR4 . Meanwhile, other environmental perturbations elicit production of an alternative substrate receptor assembling into a related E3 ligase complex. The intricate structure of GID Ant enables anticipating and ultimately binding various N-degron targeting (i.e. "N-end rule") substrate receptors, while the GID SR4 E3 forms a clamp-like structure juxtaposing substrate lysines with the ubiquitylation active site. The data reveal evolutionarily conserved GID complexes as a family of multisubunit E3 ubiquitin ligases responsive to extracellular stimuli.

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