Mechanism and engineering of a cold-adapted hetero-oligomeric ATP-phosphoribosyltransferase: implications for synthetic biology

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The fine chemicals, pharmaceutical, and agrochemical industries have an increasing interest in utilizing biocatalysts in processes. In 2010 the total global market for industrial enzymes was $ 3.3 billions, and it is predicted that by 2050, 30% of chemical business will be biocatalysis. The need to develop more selective, greener, and more cost-effective synthesis drives this shift within the industry, and it is imperative that new enzymes are developed.Bacteria that dwell in frozen environments evolved molecular adaptations to thrive in low temperature. They often express enzymes that are active at low temperature but unfold at temperatures below the melting point for their mesophilic and thermophilic counterparts. These cold-adapted enzymes attract significant interest from the biotechnology industry: they achieve useful rates of catalysis without additional thermal energy input, which reduces manufacturing costs; and they allow thermolabile products and intermediates to be rapidly synthesized under conditions that increases their stability. In spite of these advantages, our knowledge about the physical chemical properties and catalytic mechanisms of psychrophilic enzymes lags behind that about mesophilic and thermophilic ones.This project will focus on the cold-adapted bacterial enzyme ATP-phosphoribosyltransferase (ATPPRT). This enzyme catalyses the first step of histidine biosynthesis, the condensation of adenosine 5-triphosphate (ATP) and 5-phosphoribosyl-1-pyrophosphate (PRPP) to form N1-(5-phosphoribosyl)-ATP, with release of pyrophosphate This enzyme is a hetero-oligomer consisting of a catalytic subunit, HisG, and a regulatory subunit, HisZ, which confers allosteric inhibition by histidine.The overarching aim of the project is to unravel the catalytic mechanism of the reaction and harness that knowledge to expand its substrate and product scope. This will provide a tool to enable rapid diversification of sugar-nucleotide chemistry while shedding light on enzymatic catalysis at low temperature.