Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal–ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a third-generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a 3rd generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

Enzyme reactivity is often enhanced by changes in oxidation state, spin state, and metal-ligand covalency of associated metallocofactors. The development of spectroscopic methods for studying these processes coincidentally with structural rearrangements is essential for elucidating metalloenzyme mechanisms. Herein, we demonstrate the feasibility of collecting X-ray emission spectra of metalloenzyme crystals at a 3rd generation synchrotron source. In particular, we report the development of a von Hamos spectrometer for the collection of Fe Kβ emission optimized for analysis of dilute biological samples. We further showcase its application in crystals of the immunosuppressive heme-dependent enzyme indoleamine 2,3-dioxygenase. Spectra from protein crystals in different states were compared with relevant reference compounds. Complementary density functional calculations assessing covalency support our spectroscopic analysis and identify active site conformations that correlate to high- and low-spin states. These experiments validate the suitability of an X-ray emission approach for determining spin states of previously uncharacterized metalloenzyme reaction intermediates.

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 5-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 cross-link 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.

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.