Focused mutagenesis in non-catalytic cavity for improving catalytic efficiency of 3-ketosteroid-Δ1-dehydrogenase

Non-catalytic sites are commonly present in many enzymes, but how to classify and modulate such sites for activity improvement is not well-explored. Herein a non-catalytic cavity in the 3-ketosteroid-Δ1-dehydrogenase (Δ1-KstD) from Mycobacterium smegmatis (MsKstD1), which could competitively bind to substrate to form non-catalytic complex, was predicted next to substrate tunnel by the CAVER/molecular dynamics. Based on that, a rational design was performed by Focused Site-directed Iterative Saturation Mutagenesis (FSISM) for blocking the non-catalytic cavity to enhance substrate enter active site to improve the Δ1-dehydrogenation activity of MsKstD1. We obtained the best quadruple mutant (H132M\L113F\V419W\M51L) with 10-fold higher specific activity toward hydrocortisone than that of wild type enzyme. The Δ1-dehydrogenation space time yield toward hydrocortisone reached 36.0 g/L/h to produce prednisolone. This work displays how to combine focused mutagenesis with computational analysis to effectively identify and modify non-catalytic cavity to enhance enzyme activity for efficient production of Δ1-3-ketosteroid.