Nitric oxide trapping of carbon-centered radicals in an enzyme active site
Mentor 1
Nicholas Silvaggi
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
The non-proteinogenic amino acid L-enduracididine (L-End) is a component of several potent antibiotic peptides. L-End biosynthesis in Streptomyces wadayamensis begins with a unique pyridoxal-5’-phosphate (PLP) dependent L-Arg oxidase. This oxidase, MppP, uses only the PLP cofactor and molecular oxygen to catalyze the 4-electron oxidation of L-Arg to 4-hydroxy-2-ketoarginine. Hydroxylation of an unactivated methylene carbon is an unprecedented reaction for a PLP-dependent enzyme and this laboratory has set about developing a detailed understanding of the enzymatic mechanism. L-Arg reacts with PLP in the MppP active site, just as seen in other PLP-dependent enzymes. However, unlike most other PLP-dependent enzymes, MppP is able to stabilize electron-rich quinonoid intermediates for an astonishing length of time. These quinonoid intermediates are capable of 1-electron reduction of molecular oxygen to generate superoxide. These studies seek to determine whether the mechanism proceeds through a formal substrate radical, and if so, where on the substrate does the radical form. We have performed MppP reactions in the presence of a nitric oxide generator in an attempt to trap any organic radicals that form in the active site. The resulting NO adducts were detected by ion trap-time-of-flight (IT-TOF) mass spectrometry, and fragmentation patterns were used to deduce the structures.
Nitric oxide trapping of carbon-centered radicals in an enzyme active site
Union Wisconsin Room
The non-proteinogenic amino acid L-enduracididine (L-End) is a component of several potent antibiotic peptides. L-End biosynthesis in Streptomyces wadayamensis begins with a unique pyridoxal-5’-phosphate (PLP) dependent L-Arg oxidase. This oxidase, MppP, uses only the PLP cofactor and molecular oxygen to catalyze the 4-electron oxidation of L-Arg to 4-hydroxy-2-ketoarginine. Hydroxylation of an unactivated methylene carbon is an unprecedented reaction for a PLP-dependent enzyme and this laboratory has set about developing a detailed understanding of the enzymatic mechanism. L-Arg reacts with PLP in the MppP active site, just as seen in other PLP-dependent enzymes. However, unlike most other PLP-dependent enzymes, MppP is able to stabilize electron-rich quinonoid intermediates for an astonishing length of time. These quinonoid intermediates are capable of 1-electron reduction of molecular oxygen to generate superoxide. These studies seek to determine whether the mechanism proceeds through a formal substrate radical, and if so, where on the substrate does the radical form. We have performed MppP reactions in the presence of a nitric oxide generator in an attempt to trap any organic radicals that form in the active site. The resulting NO adducts were detected by ion trap-time-of-flight (IT-TOF) mass spectrometry, and fragmentation patterns were used to deduce the structures.
