Probing the active site of the Rv0045c esterase from Mycobaterium tuberculosis (580.4)

The active site of the Rv0045c esterase from Mycobaterium tuberculosis was probed with a combination of site‐directed mutagenesis and a series of substrate analogues. The thermal stability of enzyme variants was measured using differential scanning fluorimetry with Sypro Orange dye. The catalytic efficiency of the enzyme variants was measured using fluorogenic ester substrates. Variants of active site residues confirmed a classic serine catalytic triad, as well as revealing the relative importance of several other active site residues. Two active site variants (G90A and H187A) displayed increased catalytic efficiency relative to the wild type, but the corresponding double variant lacked a synergistic activation effect. A series of other H187 variants revealed that further fine‐tuning of enzymatic activity was possible. Additionally, the relative catalytic efficiencies of the hydrolysis of a series of flourogenic ester substrates revealed structure activity relationships, in which short chain, unbranched, hydrogen bond accepting esters displayed maximal activity. Grant Funding Source : Supported by the National Science Foundation