Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution

Abstract

In human pathogens, the sulphate assimilation pathway provides reduced sulphur for biosynthesis of essential metabolites including cysteine and low-molecular weight thiol compounds. Sulfonucleotide reductases (SRs) catalyze the first committed step of sulphate reduction. In this reaction, activated sulphate in the form of adenosine-5′-phosphosulphate (APS) or 3′-phosphoadenosine 5′-phosphosulphate (PAPS) is reduced to sulphite. Gene knockouts, transcriptomic and proteomic data establish the importance of SRs in oxidative stress-inducible antimicrobial resistance mechanisms. In previous work, we have 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 have used directed evolution to rapidly identify functional sites in PAPS reductase (PAPR). Four new regions are 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.