<div> <div> <div> <p>Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin. </p> </div> </div> </div>

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is five-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether crosslink on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

<div> <div> <div> <p>Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics (GPAs). The latter are canonical GPAs with broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridium difficile. Here, we investigated the three-dimensional structures and functional consequences for both molecules. Equilibrium binding studies showed tight binding by keratinimicin A, but not keratinicyclin B, to the peptidoglycan terminus. Using protein crystallography methods, we solved the X-ray crystal structures of both GPAs, which, in conjunction with DFT calculations, indicate that the inability of keratinicyclin B to bind the peptidoglycan is governed by steric factors. Keratinicyclin B, therefore, interferes with an alternative target to inhibit C. difficile growth, a conclusion confirmed by checkerboard analysis that revealed synergistic activity with vancomycin. Our results set the stage for identifying the molecular target of keratinicyclins and for exploring their therapeutic utility in combination with vancomycin. </p> </div> </div> </div>