Initial investigations of C4a-(hydro)peroxyflavin intermediate formation by dibenzothiophene monooxygenase.

Dibenzothiophene monooxygenase is the initiating enzyme in the Rhodococcus 4S biodesulfurization pathway. A member of the Class D flavin monooxygenases, it uses FMN to activate molecular oxygen for oxygenation of the substrate, dibenzothiophene. Here, we have used stopped-flow spectrophotometry to show that DszC forms a peroxyflavin intermediate in the absence of substrate. Mutagenesis of Ser163 and His391 to Ala appears to decrease the binding affinity for reduced FMN and eliminates the enzyme's ability to stabilize the peroxyflavin intermediate.

Origins of glycan selectivity in streptococcal Siglec-like adhesins suggest mechanisms of receptor adaptation.

Bacterial binding to host receptors underlies both commensalism and pathogenesis. Many streptococci adhere to protein-attached carbohydrates expressed on cell surfaces using Siglec-like binding regions (SLBRs). The precise glycan repertoire recognized may dictate whether the organism is a strict commensal versus a pathogen. However, it is currently not clear what drives receptor selectivity. Here, we use five representative SLBRs and identify regions of the receptor binding site that are hypervariable in sequence and structure. We show that these regions control the identity of the preferred carbohydrate ligand using chimeragenesis and single amino acid substitutions. We further evaluate how the identity of the preferred ligand affects the interaction with glycoprotein receptors in human saliva and plasma samples. As point mutations can change the preferred human receptor, these studies suggest how streptococci may adapt to changes in the environmental glycan repertoire.

Fragment-Based Discovery of Small Molecules Bound to T-Cell Immunoglobulin and Mucin Domain-Containing Molecule 3 (TIM-3).

T-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3; HAVCR2) has emerged as an attractive immune checkpoint target for cancer immunotherapy. TIM-3 is a negative regulator of the systemic immune response to cancer and is expressed on several dysfunctional, or exhausted, immune cell subsets. Upregulation of TIM-3 is associated with tumor progression, poor survival rates, and acquired resistance to antibody-based immunotherapies in the clinic. Despite the potential advantages of small-molecule inhibitors over antibodies, the discovery of small-molecule inhibitors has lagged behind that of antibody therapeutics. Here, we describe the discovery of high-affinity small-molecule ligands for TIM-3 through an NMR-based fragment screen and structure-based lead optimization. These compounds represent useful tools to further study the biology of TIM-3 immune modulation in cancer and serve as a potentially useful starting point toward the discovery of TIM-3-targeted therapeutics.