Molecular Basis for Peptide Nitration by a Novel Cytochrome P450 Enzyme in RiPP Biosynthesis

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Cytochrome P450 enzymes (CYPs) are crucial in biosynthesis, with RufO uniquely involved in nitrated peptide production.
• Understanding RufO's mechanism is key to advancing knowledge of nitrating enzymes and ribosomally synthesized and post-translationally modified peptides (RiPPs).
• The precise molecular basis for RufO's substrate recognition and nitration of MRYLH remains largely unelucidated.

Purpose of the Study:

• To elucidate the molecular basis of peptide binding and heme-based nitration in the cytochrome P450 enzyme RufO.
• To investigate RufO's substrate specificity and catalytic mechanisms in the context of RiPP biosynthesis.

Main Methods:

• Employed a multidisciplinary approach combining spectroscopic, kinetic, and structural techniques.
• Determined a high-resolution crystal structure (1.51 Å) of RufO.
• Conducted transient kinetic studies to analyze oxygen and nitric oxide binding.

Main Results:

• Peptide binding to RufO is endothermic with a dissociation constant of 0.78 μM, showing minimal heme perturbation.
• Substrate binding induces significant conformational changes in RufO's distal pocket, notably involving interactions with peptide residues Arg-2 and His-5.
• Kinetic data revealed sequential O2 and •NO binding, forming a ferric-superoxo intermediate critical for nitration.

Conclusions:

• RufO exhibits unique substrate binding and catalytic features distinct from other characterized nitrating CYPs like TxtE.
• An extended hydrogen-bonding network constraining His-5 is identified as a key structural element for RufO's specific nitration activity.
• This research provides critical insights into the enzymatic mechanisms underlying nitration reactions within RiPP biosynthesis pathways.