The creation of artificial enzymes to catalyze desired reactions is a major goal of computational protein design. However, de novo enzymes display low catalytic efficiencies, requiring the introduction of activity-enhancing active site and distal mutations through directed evolution. A better understanding of how mutations introduced by directed evolution contribute to increased enzymatic activity will guide the development of design methods such that efficient enzymes can be designed de novo. Here, we evaluate the structural, functional, and dynamical impacts of active site and distal mutations introduced by directed evolution of the de novo retro-aldolase RA95, an enzyme that presents an important case study in enzyme design due to the significant structural remodelling that was observed during evolution. We observe that the variant RA95-Core, containing only active site mutations introduced by directed evolution, displays activity within one order of magnitude of the fully evolved variant. This suggests that computational enzyme design methods can be improved to create much more efficient enzymes than what was previously achieved in RA95. However, structural changes induced by distal mutations prevent computational recapitulation of the evolved active site on the original design template, indicating that the optimized active site identified through directed evolution could not have been designed de novo using current design methodologies. We suggest strategies for the incorporation of backbone remodelling into design procedures that would allow recapitulation of the evolved retro-aldolase active site, as well as the de novo design of highly efficient enzymes without the need for optimization by directed evolution.

Structural biology uncovers life's secrets by studying protein structures via techniques like X-ray crystallography. This knowledge drives advancements in protein engineering for the improvement of human lives. Yet, obtaining high-quality crystals in X-ray crystallography is challenging. To overcome this, we used Translocation ETS Leukemia protein Sterile Alpha Motif domain (TELSAM), a promising polymer-forming crystallization chaperone (PFCC), to enhance protein crystallization. Human thirty-eight-negative kinase-1 (TNK1), a key player in cancer progression, possess a ubiquitin association (UBA) domain that binds polyubiquitin and regulates TNK1 activity and stability. Although sequence analysis hints at an unconventional TNK1 UBA domain architecture, its molecular structure lacks experimental validation. To gain insight into TNK1 regulation, we fused the UBA domain to the 1TEL crystallization chaperone and obtained crystals diffracting as far as 1.53 Ã…. 1TEL enabled solution of the X-ray phases. GG and GSGG linkers allowed the UBA to reproducibly find a productive binding mode against its 1TEL polymer and to crystallize at protein concentrations as low as 0.1 mg/mL. Our findings support a TELSAM fusion crystallization mechanism, highlighting fewer crystal contacts compared to traditional crystals. Both modeling and experimental validation indicate that the UBA domain exhibits selectivity towards polyubiquitin chain length and linkages. Radical S-adenosylmethionine (SAM) enzymes catalyze various radical-mediated substrate transformations. Despite the growing interest of computational enzyme design in industrial small molecule synthesis, radical SAM enzymes remain relatively unexplored. We used PyRosetta to leverage hydrogen bonding design (hbDes) and hydrophobic interaction design (hpDes) to enable a radical cyclization reaction on our selected substrate. Although the purified enzymes demonstrated activation potential with a reducing agent, enzymatic assays failed to exhibit activity against the reactant. To obtain successful results, addressing additional questions and issues is required, which may involve the implementation of machine learning.

Biocatalysis represents an attractive route to the production of various compounds which are difficult or impossible to synthesise and isolate using traditional chemical synthesis. In particular, the production of chiral molecules is a function ideally suited to biocatalysis, due to the natural stereospecificity of enzymes. The synthesis of such chiral molecules is essential in the production of pharmaceuticals, additives for the food and drinks industry and the creation of specialist polymers. CYP105AS1, isolated from Amycolatopsis orientalis, is a cytochrome P450 enzyme which produces the inactive 6-epi-pravastatin of the blockbuster anti-cholesterol drug pravastatin. Previous directed evolution efforts have engineered this enzyme to produce a five-point mutant, known as P450prava, which partially reversed the stereospecificity of the enzyme to produce a majority pravastatin product mixture. This thesis details work to use structure-led engineering approaches to redesign the active site of P450prava to introduce stringent stereospecificity. A combinatorial approach of manual and computational rational design was pursued, leading to the creation of a novel T95F/V180M double mutant of P450prava. This double mutant was found to have successfully eliminated the unwanted 6-epi-pravastatin enantiomer from the product mix, leaving a pure pravastatin product. P450prava was also shown to bind and hydroxylate other statin substrate molecules, demonstrating its versatility in the production of drug metabolites and other high-value oxyfunctionalised molecules. This property, along with its proven tolerance of significant active site engineering efforts, demonstrates the viability of the P450prava as a platform for the creation of novel biocatalysts for the production of various hydroxylated products from diverse substrate molecules.