Understanding the Thermodynamic and Kinetic Properties of the Thermophilic Oxoglutarate Carboxylase BC domain to Increase Activity for use in Biofuel Crops

Abstract

For many biofuel crops, carbon fixation is limiting –making the production of these alternative fuels economically unviable. The first green revolution has led to significant advances in crop yields in the past half century. Crop yield is now mostly limited by plant biology. To overcome this limitation, a condensed reverse TCA (crTCA) cycle has been developed in the workhorse species camelina that rapidly fixes atmospheric carbon. The biotin‐dependent carboxylase Oxoglutarate Carboxylase (OGC) represents the slow step of the crTCA cycle engineered in camelina. OGC –a thermophile –is 500‐fold less active at mesophilic temperatures than at its optimal 70 °C. Engineering OGC to preserve high activity at mesophilic temperatures presents an opportunity to further improve the rate of carbon fixation in the crTCA pathway, and possibly increase yields in oil crops –reducing the cost of biofuels.Biotin‐dependent carboxylases such as OGC are a ubiquitously found family of proteins that consist of three domains: The Biotin Carboxylase (BC) domain, Biotin Carboxyl Carrier Protein (BCCP), and the Carboxy Transferase (CT) domain. We choose to analyze the BC domain as previous studies suggest the rate limiting step of the enzyme to result from 1) conformational changes associated with lid closure in the domain or 2) deprotonation of a species within the active site. We report kinetic and thermodynamic ATPase activity measurements of the thermophilic OGC BC domain measured via discontinuous assay coupled with direct quantitation of ADP production via HPLC. ATP concentration dependent activity was observed at temperatures ranging from 48 °C to 76°C, with kcat at 76 °C being 93 min−1. A subsequent Eyring plot was generated to measure the free energy of the transition state of OGC BC. Along with this, kinetic deuterium isotope effect experiments are being performed to measure energetics of deprotonation in the active site. Lid motions are also currently being measured with FRET. These studies will aid in the identification of mutations that may increase the activity of the BC domain at mesophilic temperatures and facilitate the use of OGC to increase biofuel crop yields.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.