Biochemical characterization of Fsa16295Glu from "Fervidibacter sacchari," the first hyperthermophilic GH50 with ß-1,3-endoglucanase activity and founding member of the subfamily GH50_3.

The aerobic hyperthermophile "Fervidibacter sacchari" catabolizes diverse polysaccharides and is the only cultivated member of the class "Fervidibacteria" within the phylum Armatimonadota. It encodes 117 putative glycoside hydrolases (GHs), including two from GH family 50 (GH50). In this study, we expressed, purified, and functionally characterized one of these GH50 enzymes, Fsa16295Glu. We show that Fsa16295Glu is a ß-1,3-endoglucanase with optimal activity on carboxymethyl curdlan (CM-curdlan) and only weak agarase activity, despite most GH50 enzymes being described as ß-agarases. The purified enzyme has a wide temperature range of 4-95°C (optimal 80°C), making it the first characterized hyperthermophilic representative of GH50. The enzyme is also active at a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa16295Glu possesses a relatively high kcat/KM of 1.82 × 107 s-1 M-1 with CM-curdlan and degrades CM-curdlan nearly completely to sugar monomers, indicating preferential hydrolysis of glucans containing ß-1,3 linkages. Finally, a phylogenetic analysis of Fsa16295Glu and all other GH50 enzymes revealed that Fsa16295Glu is distant from other characterized enzymes but phylogenetically related to enzymes from thermophilic archaea that were likely acquired horizontally from "Fervidibacteria." Given its functional and phylogenetic novelty, we propose that Fsa16295Glu represents a new enzyme subfamily, GH50_3.

Responsive Antibody Conjugates Enable Quantitative Determination of Intracellular Bond Degradation Rate.

Degradable crosslinkers that respond to intracellular biological stimuli are a critical component of many drug delivery systems. With numerous stimuli-responsive drug delivery systems in development, it is important to quantitatively study their intracellular processing. Herein we report a framework for quantifying the rate of intracellular bond degradation in the endocytic pathway. Toward this end, we devised and synthesized a reduction-sensitive FRET-based crosslinker that can be readily conjugated to a variety of targeting ligands. This crosslinker was conjugated to trastuzumab, a humanized monoclonal antibody against the HER2 receptor. We developed a model based on mass-action kinetics to describe the intracellular processing of this conjugate. The kinetic model was developed in conjunction with live-cell experiments to extract the rate constant for intracellular disulfide bond degradation. This framework may be applied to other endocytosis pathways, bond types, and cell types to quantify this fundamental degradation rate parameter.