NSF-DBT: Structural and Biophysical Analysis of Evolved Fe(II)/2-Ketoglutarate-Dependent Azidases to Understand and Expand the Substrate Scope of Enzymatic C-H Functionalization

Indiana University, Bloomington IN

Investigators

Abstract

Enzymes promote reactions by binding the substrate, catalyzing the reaction, and releasing the product. Each of these activities depends on the three-dimensional shape of the enzyme. Efforts to enhance the overall productivity of an enzyme usually focus on increasing the rate of the reaction in the active site. However, altering the shape to improve one aspect of its activity can change the shape in other parts of the enzyme. This often leads to situations where a single change improves the rate of reaction but decreases the rate of binding of reactants or release of products. There could be no net improvement or even a decrease in overall productivity. The challenge is even greater when the goal of the enzyme engineering is to have it catalyze a related but new reaction, with different reactants. Traditionally, this is attempted by presenting the enzyme with a single representative novel substrate and attempting to modify the enzyme to bind it and catalyze a new reaction. The central concept of the project is that simultaneously evolving both the enzyme and the substrate might be a more effective means to achieve improved performance. Enzymes will be exposed to a variety of nitrogen-containing substrates to develop an enzyme that will convert carbon-hydrogen (C-H) bonds directly into carbon-nitrogen (C-N) bonds. To encourage the development of the biomanufacturing workforce, workshops and online courses on enzyme engineering will be developed. These will be offered through both Indiana University and through the Centre for Continuing Education at IIT Hyderabad, India. An engineered Fe(II)- and α-ketoglutarate dependent enzyme termed SadX can catalyze site-selective C-H azidation and carbamoylation. Directed evolution improved SadX azidase activity via mutations throughout its structure. It is unclear how these mutations affected activity. The resulting variants exhibited limited substrate scope. The project will attempt to provide fundamental insight into non-native reactivity and selectivity of SadX variants and expand the scope of enzymatic C-H functionalization to incorporate nitrogen-containing functionality. The Eerappa group will study enzymes in the azidase lineage using solution biophysical methods, X-ray crystallography, computational modeling, and density functional theory (DFT)-based quantum mechanical analysis to gain insight into how mutations in the evolved variants lead to changes in active site structure and substrate binding. The Lewis group will then use computational modeling to design substrates that contain features found to be essential for effective C-H functionalization while modifying non-essential features to gradually expand substrate scope. At the same time, structure- and MD simulation-guided mutagenesis will be used to further evolve variants with improved activity on modified substrates. Simultaneous evolution of both substrate and enzyme structure might enable more efficient expansion of substrate scope than protein engineering aimed at forcing the enzyme to adapt to a particular substrate. The proposed research will provide insight into how engineered Fe(II)- and α-ketoglutarate dependent enzymes can accommodate diverse anionic ligands to enable non-native C-H functionalization reactions. It is anticipated that the proposed studies will greatly improve the ability to further engineer non-native catalysis. Validating the efficacy of the proposed approach to simultaneous substrate modification and enzyme evolution for expanding enzyme scope could also be used to improve the generality of other enzymes for biocatalysis. This project involves a collaboration between researchers from the United State and India. It is jointly supported by the US National Science Foundation and the Department of Biotechnology of the Government of India (NSF-DBT). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.