Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution. (12th August 2016) - Paritala, Hanumantharao
Abstract: In human pathogens, the sulfate assimilation pathway provides reduced sulfur for biosynthesis of essential metabolites, including cysteine and low‐molecular‐weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulfate reduction. In this reaction, activated sulfate in the form of adenosine‐5′‐phosphosulfate (APS) or 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) is reduced to sulfite. Gene knockout, transcriptomic and proteomic data have established the importance of SRs in oxidative stress‐inducible antimicrobial resistance mechanisms. In previous work, we focused on rational and high‐throughput design of small‐molecule inhibitors that target the active site of SRs. However, another critical goal is to discover functionally important regions in SRs beyond the traditional active site. As an alternative to conservation analysis, we used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions were discovered that are essential to PAPR function and lie outside the substrate binding pocket. Our results highlight the use of directed evolution as a tool to rapidly discover functionally important sites in proteins. Abstract : Evolution and discovery : We used directed evolution to rapidly identify new functional sites in the sulfur metabolism enzyme PAPS reductase (PAPR). These regions might have regulatory or allosteric functions and could lay the foundation for the development of molecules that target regions of PAPR, are distinct from the active site and are smaller than most natural protein–protein interfaces.