DEAllAct · Dissecting and Engineering Allosteric Activation in a Biosynthetic Enzyme

Dissecting and Engineering Allosteric Activation in a Biosynthetic Enzyme

Enzymes are biological catalysts that speed up the rate of a specific chemical reaction in the cell, performing important functions and supporting life. Enzymes can be allosterically modulated, i.e. altering their reaction rate and/or substrate affinity upon binding to or mutation of a site remote from the active site of the enzyme. These processes are fundamental regulatory mechanisms of biochemical reactions that often involve a complex network of interactions. The enzyme ATP phosphoribosyltransferase (ATPPRT) from the cold-adapted bacterium Psychrobacter arcticus, is responsible for the first and flux-controlling step in histidine biosynthesis and is subject to complex allosteric control. ATPPRT is constituted of two parts, the protein possessing the catalytic domains (HisGs) and a catalytically inactive regulatory (HisZ). HisZ has a dual function: it allosterically enhances catalysis by HisGs, and it binds histidine and mediates allosteric inhibition, making it a model system for understanding the allosteric regulation of catalysis. Overcoming histidine inhibition in ATPPRT is key in synthetic biology efforts toward histidine production in bacteria. The DEAllAct project aimed to understand the molecular details of the catalytic and allosteric mechanism of ATPPRT enzyme to predict and test mutations at the protein-protein interface that enhance the catalytic activity of the protein containing the catalytic domains (HisGs) without the regulatory domain (HisZ) by mimicking its allosteric activation. The project also aimed to explore the design of a computational tool to dissect allostery by combining computational simulations and biophysical experimental studies that can be used to predict specific mutations at the protein-protein interface of allosterically regulated complexes. Conclusions of the action: 1. Successful identification of interaction networks that lead to allosteric activation of HisGs upon binding to the regulatory protein. 2. Demonstrated the involvement of two key residues at the binding domain that can rescue each other for the catalytic activation of the enzyme upon binding of the regulatory protein. 3. Developed a tool capable of identifying residues of key importance for binding interactions between the enzyme and its regulatory protein. 4. Identified and computationally validated 5 hotspots at the binding interface of HisGs that can potentially enhance its stand-alone activity.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз