Figure 6
Improved stereoselectivity of the E144/W242 double mutant toward (2S,3R,4S)-4-HIL. (a) Binding model of (2S,3R,4S)-4-HIL to the active site of HILDH. (2S,3R,4S)-4-HIL, HILDH and NADH are shown in yellow, slate and white sticks. Distances in angstroms are denoted by dotted lines. (b) HPLC chromatogram (upper) and bar graph (lower) of 4-HIL stereoisomers produced by the wild type (WT) and the double mutants of E144 and W242. “No enzyme” indicates the reaction mixtures without HILDH. The reaction mixtures were derivatized with GITC and detected by HPLC. Dotted line in bar graph divides products into 4S forms (below the dotted line) and 4R forms (above the dotted line) of 4-HIL. (c) F o - F c electron density omit map (σ = 1.0) of K144 and Q242 of HILDHE144K/W242Q-NADH- succinate complex. Succinate, HILDH and NADH are shown in orange, slate and white sticks, respectively. Red spheres represent water molecules. Dotted lines show potential hydrogen bonds or salt bridges. Distances are denoted in angstroms. (d) F o - F c electron density omit map (σ = 1.0) of succinate bounded in the structure of HILDHE144K/W242Q-NADH- succinate complex. Succinate, HILDH and NADH are shown in orange, slate and white sticks, respectively. Dotted lines show hydrogen bonds or salt bridges. (e) The binding model of (2S,3R,4S)-4-HIL to HILDHE144K/W242Q constructed by docking simulation. K144 and Q242 are shown in pink sticks. (2S,3R,4S)-4-HIL, HILDH and NADH are shown in yellow, slate and white sticks, respectively. Red spheres represent water molecules. Dotted lines show potential hydrogen bonds or salt bridges. Distances are denoted in angstroms.
