Metabolic inhibitors based on rare bacterial monosaccharide scaffolds interfere with bacterial glycan biosynthesis in a bacteria-selective manner.
Glycosidic bond formation is a continual challenge for practitioners. Aiming to enhance the reproducibility and efficiency of oligosaccharide synthesis, we studied the relationship between glycosyl donor activation and reaction temperature. A novel semi‐automated assay revealed diverse responses of members of a panel of thioglycosides to activation at various temperatures. The patterns of protecting groups and the thiol aglycon combine to cause remarkable differences in temperature sensitivity among glycosyl donor building blocks. We introduce the concept of donor activation temperature to capture experimental insights, reasoning that glycosylations performed below this reference temperature evade deleterious side reactions. Activation temperatures enable a simplified temperature treatment and facilitate optimization of glycosyl donor usage. Isothermal glycosylation below the activation temperature halved the equivalents of building block required in comparison to the standard “ramp” regime used in solution‐ and solid‐phase oligosaccharide synthesis to‐date.
Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin–Benson–Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
CRISPR–Cas adaptive immune systems capture DNA fragments from invading mobile genetic elements and integrate them into the host genome to provide a template for RNA-guided immunity1. CRISPR systems maintain genome integrity and avoid autoimmunity by distinguishing between self and non-self, a process for which the CRISPR/Cas1–Cas2 integrase is necessary but not sufficient2–5. In some microorganisms, the Cas4 endonuclease assists CRISPR adaptation6,7, but many CRISPR–Cas systems lack Cas48. Here we show here that an elegant alternative pathway in a type I-E system uses an internal DnaQ-like exonuclease (DEDDh) to select and process DNA for integration using the protospacer adjacent motif (PAM). The natural Cas1–Cas2/exonuclease fusion (trimmer-integrase) catalyses coordinated DNA capture, trimming and integration. Five cryo-electron microscopy structures of the CRISPR trimmer-integrase, visualized both before and during DNA integration, show how asymmetric processing generates size-defined, PAM-containing substrates. Before genome integration, the PAM sequence is released by Cas1 and cleaved by the exonuclease, marking inserted DNA as self and preventing aberrant CRISPR targeting of the host. Together, these data support a model in which CRISPR systems lacking Cas4 use fused or recruited9,10 exonucleases for faithful acquisition of new CRISPR immune sequences.
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