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