Direct Detection of the ��-Carbon Radical Intermediate Formed by OspD: Mechanistic Insights into Radical -Adenosyl-l-methionine Peptide Epimerization.
William G Walls, Anna L Vagstad, Tyler Delridge, J��rn Piel, William E Broderick, Joan B Broderick
Author Information
William G Walls: Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, United States. ORCID
Anna L Vagstad: Institute of Microbiology, Eidgen��ssische Technische Hochschule (ETH) Z��rich, Vladimir-Prelog-Weg 4, Z��rich 8093, Switzerland. ORCID
Tyler Delridge: Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, United States.
J��rn Piel: Institute of Microbiology, Eidgen��ssische Technische Hochschule (ETH) Z��rich, Vladimir-Prelog-Weg 4, Z��rich 8093, Switzerland.
William E Broderick: Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, United States. ORCID
Joan B Broderick: Department of Chemistry & Biochemistry, Montana State University, Bozeman, Montana 59717, United States. ORCID
OspD is a radical -adenosyl-l-methionine (SAM) peptide epimerase that converts an isoleucine (Ile) and valine (Val) of the OspA substrate to d-amino acids during biosynthesis of the ribosomally synthesized and post-translationally modified peptide (RiPP) natural product landornamide A. OspD is proposed to carry out this reaction via ��-carbon (C��) H-atom abstraction to form a peptidyl C�� radical that is stereospecifically quenched by hydrogen atom transfer (HAT) from a conserved cysteine (Cys). Here we use site-directed mutagenesis, freeze-quench trapping, isotopic labeling, and electron paramagnetic resonance (EPR) spectroscopy to provide new insights into the OspD catalytic mechanism including the direct observation of the substrate peptide C�� radical intermediate. The putative quenching Cys334 was changed to serine to generate an OspD C334S variant impaired in HAT quenching. The reaction of reduced OspD C334S with SAM and OspA freeze-quenched at 15 s exhibits a doublet EPR signal characteristic of a C�� radical coupled to a single ��-H. Using isotopologues of OspA deuterated at either Ile or Val, or both Ile and Val, reveals that the initial C�� radical intermediate forms exclusively on the Ile of OspA. Time-dependent freeze quench coupled with EPR spectroscopy provided evidence for loss of the Ile C�� radical concomitant with gain of a Val C�� radical, directly demonstrating the N-to-C directionality of epimerization by OspD. These results provide direct evidence for the aforementioned OspD-catalyzed peptide epimerization mechanism via a central C�� radical intermediate during RiPP maturation of OspA, a mechanism that may extend to other proteusin peptide epimerases.
References
Angew Chem Int Ed Engl. 2020 Jul 13;59(29):11763-11768
[PMID: 32163654]
