Fig. 2
From: Deciphering the regulatory and catalytic mechanisms of an unusual SAM-dependent enzyme
Structure of LepI in complex with MTA and SAM. a Architecture of the LepI dimers in complex with SAM and MTA. LepI adopts a SAM-dependent MT fold. Small molecules are indicated as spheres in the cavity of LepI structure. b The cavity comprises two sites: a SAM site and a substrate site. The close-up stereo view of the mimic substrate MTA and SAM-binding site indicates that the SAM (indicated by the red circle) site is independent of the substrate site (indicated by the green rectangle). c The 2Fo–Fc electron densities for MTA (colored cyan) and SAM (colored pink) at 1σ and 2σ, respectively. A close-up view of the detailed interaction between SAM and LepI is shown; gray dashed lines indicate the hydrogen bonds, and the blue dashed line indicates the π–π interaction. d Analytic gel-filtration of purified LepI-Δ15. Three peaks appear, representing the formation of LepI monomer, dimer, and tetramer according to the standard protein marker. A representative image from three replicate experiments is shown. e Enzymatic activity of LepI-Δ15 Fr1-Fr3 compared with wild-type (WT) LepI determined through retro-rearrangement assay with triplicate measurement. (Data represent the mean ± s.d.) The Fr3 monomer indicated in the gel-filtration assay is almost inactive, whereas both Fr1 and Fr2 still have full activity. f Limited proteolysis of LepI in the presence of SAM, SAH, or MTA at gradient concentrations of trypsin. The proteolytic fragments were detected by SDS-PAGE and Coomassie staining
