Extended Data Fig. 3: Activity assays on the wild-type CghA and its mutants to examine the roles of the active-site residues.

The in vitro analyses were performed using the substrate analogue 8 for the formation of the endo 9 and exo 10 adducts. Detailed reaction conditions are described in Methods unless otherwise specified below. a, HPLC profiles of the reaction mixtures containing 8 as a substrate and (i) the S65N mutant; (ii) the N82A mutant; (iii) the H94A mutant; (iv) the K352A mutant; (v) the N364A mutant; (vi) the wild-type CghA; (vii) buffer, as a background reaction showing the conversion of 8 to 9 and 10 after 10 minutes of incubation; (viii) the authentic reference of 9; and (ix) the authentic reference of 10. The reactions were monitored at 288 nm. N-Boc-L-tryptophan methyl ester was used as an internal standard (IS) throughout the study. See Methods for details. Peaks denoted by asterisks are impurities from the chemical synthesis of 8. b, Hydrogen-bonding interaction between the bound ligand P-1 and the S65 and N82 side chain groups. Two water molecules also link the S65 and N82 side chain groups through hydrogen bonds.