Direct Detection of the α-Carbon Radical Intermediate Formed by OspD: Mechanistic Insights into Radical S-Adenosyl-l-methionine Peptide Epimerization.

OspD is a radical S-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 α-carbon (Cα) H-atom abstraction to form a peptidyl Cα 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 Cα 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 Cα radical coupled to a single ß-H. Using isotopologues of OspA deuterated at either Ile or Val, or both Ile and Val, reveals that the initial Cα radical intermediate forms exclusively on the Ile of OspA. Time-dependent freeze quench coupled with EPR spectroscopy provided evidence for loss of the Ile Cα radical concomitant with gain of a Val Cα 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 Cα radical intermediate during RiPP maturation of OspA, a mechanism that may extend to other proteusin peptide epimerases.

ENDOR Spectroscopy Reveals the "Free" 5'-Deoxyadenosyl Radical in a Radical SAM Enzyme Active Site Actually is Chaperoned by Close Interaction with the Methionine-Bound [4Fe-4S]2+ Cluster.

1/2H and 13C hyperfine coupling constants to 5'-deoxyadenosyl (5'-dAdo•) radical trapped within the active site of the radical S-adenosyl-l-methionine (SAM) enzyme, pyruvate formate lyase-activating enzyme (PFL-AE), both in the absence of substrate and the presence of a reactive peptide-model of the PFL substrate, are completely characteristic of a classical organic free radical whose unpaired electron is localized in the 2pπ orbital of the sp2 C5'-carbon (J. Am. Chem. Soc. 2019, 141, 12139-12146). However, prior electron-nuclear double resonance (ENDOR) measurements had indicated that this 5'-dAdo• free radical is never truly "free": tight van der Waals contact with its target partners and active-site residues guide it in carrying out the exquisitely precise, regioselective reactions that are hallmarks of RS enzymes. Here, our understanding of how the active site chaperones 5'-dAdo• is extended through the finding that this apparently unexceptional organic free radical has an anomalous g-tensor and exhibits significant 57Fe, 13C, 15N, and 2H hyperfine couplings to the adjacent, isotopically labeled, methionine-bound [4Fe-4S]2+ cluster cogenerated with 5'-dAdo• during homolytic cleavage of cluster-bound SAM. The origin of the 57Fe couplings through nonbonded radical-cluster contact is illuminated by a formal exchange-coupling model and broken symmetry-density functional theory computations. Incorporation of ENDOR-derived distances from C5'(dAdo•) to labeled-methionine as structural constraints yields a model for active-site positioning of 5'-dAdo• with a short, nonbonded C5'-Fe distance (∼3 Å). This distance involves substantial motion of 5'-dAdo• toward the unique Fe of the [4Fe-4S]2+ cluster upon S-C(5') bond-cleavage, plausibly an initial step toward formation of the Fe-C5' bond of the organometallic complex, Ω, the central intermediate in catalysis by radical-SAM enzymes.