Fig. 4.

Dissecting the two-step 4e^- reduction mechanism: (A) Stereoimage shows the cofactor NADPH (magenta) and substrate valeryl-FTAA-ppant (red) modeled in the R domain. A movement of the loop, S153-F166 (green) would provide a platform for binding of the NADPH followed by valeryl-FTAA-ppant. The extrusion of the aldehyde formed after first reduction might facilitate the exchange of oxidized cofactor NADP⁺ for NADPH. (B) Structural parameters determined for R_GPL and R_NRP along with NADPH using Guinier approximation and indirect Fourier transformation of SAXS data. Also shown is an illustrative model demonstrating loop movement along with compaction of N- and C-terminal domains in open and closed conformations. (C) NADPH binding to R_GPL and Y227F, K231A, and T192A mutants as determined by intrinsic NADPH fluorescence (Left) and analysis of NADPH consumption in spectrophotometric assays of these mutants and WT R_GPL using valeryl-FTA-Alaninal substrate (Right). (WT R_GPL K_d for NADPH was calculated as 4.64 ± 0.94 μM.) (D) Proposed mechanism of R domain-catalyzed reduction of lipopeptidyl thioester substrate.