Natural Products have long been used as drugs and drug templates for many different therapies, ranging from antibiotics to chemotherapy. Microorganisms use a large suite of enzymes in order to create novel compounds that feature interesting chemical characteristics that are often difficult to reproduce using traditional synthetic chemistry. This project attempts to bend one such enzyme to our will to create new natural product-based compounds that will allow for more flexibility in drug design. This project is focused on engineering new products from methionine adenosyltransferases (MATs), which catalyze the formation of S-adenosylmethionine, a crucial methyl donor, from methionine and ATP. Our goal is to engineer different R-groups instead of the canonical methyl, which would facilitate engineering of natural product derived compounds with enhanced activity. To this end, we have solved the crystal structure of a thermostable MAT and mutagenized it with the goal of opening up the binding pocket to accept new and interesting substrates.

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Bacillus proteases commonly exhibit remarkably reduced activity under cold conditions. Herein, we employed a tailored combination of a loop engineering strategy and iterative saturation mutagenesis method to engineer two loops for substrate binding at the entrance of the substrate tunnel of a protease (bcPRO) from Bacillus clausii to improve its activity under cold conditions. The variant MT6 (G95P/A96D/S99W/S101T/P127S/S126T) exhibited an 18.3-fold greater catalytic efficiency than the wild-type (WT) variant at 10 °C. Molecular dynamics simulations and dynamic tunnel analysis indicated that the introduced mutations extended the substrate-binding pocket volume and facilitated extra interactions with the substrate, promoting catalysis through binding in a more favorable conformation. This study provides insights and strategies relevant to improving the activities of proteases and supplies a novel protease with enhanced activity under cold conditions for the food industry to maintain the initial flavor and color of food and reduce energy consumption.