Structural and mechanistic basis for RiPP epimerization by a radical SAM enzyme.

Kubiak X, Polsinelli I, Chavas LMG, Fyfe CD, Guillot A, Fradale L, Brewee C, Grimaldi S, Gerbaud G, Thureau A, Legrand P, Berteau O, Benjdia A, Nat Chem Biol (2023) Europe PMC

SASDRS7 – Radical SAM enzyme peptide epimerase in the presence of SAM (S-adenosyl-L-methionine)

Putative peptide biosynthesis protein YydG
MWexperimental 73 kDa
MWexpected 80 kDa
VPorod 115 nm3
log I(s) 6.65×10-2 6.65×10-3 6.65×10-4 6.65×10-5
Putative peptide biosynthesis protein YydG small angle scattering data  s, nm-1
ln I(s)
Putative peptide biosynthesis protein YydG Guinier plot ln 6.66×10-2 Rg: 2.9 nm 0 (2.9 nm)-2 s2
(sRg)2I(s)/I(0)
Putative peptide biosynthesis protein YydG Kratky plot 1.104 0 √3 sRg
p(r)
Putative peptide biosynthesis protein YydG pair distance distribution function Rg: 2.9 nm 0 Dmax: 11.8 nm

Data validation

Fits and models

log I(s)
 s, nm-1
Putative peptide biosynthesis protein YydG DADIMODO model
Synchrotron SAXS data from solutions of radical SAM enzyme peptide epimerase with SAM in 25 mM HEPES, 150 mM NaCl, 1 mM DTT, pH 7.5 were collected on the SWING beam line at SOLEIL (Saint-Aubin, France) using a Eiger 4M detector at a sample-detector distance of 2 m and at a wavelength of λ = 0.1033 nm (I(s) vs s, where s = 4πsinθ/λ, and 2θ is the scattering angle). In-line size-exclusion chromatography (SEC) SAS was employed. The SEC parameters were as follows: A 50.00 μl sample at 5 mg/ml was injected at a 0.25 ml/min flow rate onto a GE Superdex 200 5/150 column at 20°C. 600 successive 0.990 second frames were collected through the entire SEC elution. The data were normalized to the intensity of the transmitted beam and radially averaged; the scattering of the solvent-blank was subtracted from the SEC-peak sample frames.

Putative peptide biosynthesis protein YydG (YydG)
Mol. type   Protein
Organism   Bacillus subtilis (strain 168)
Olig. state   Dimer
Mon. MW   39.9 kDa
 
UniProt   Q45595 (1-319)
Sequence   FASTA