title: Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution.
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.
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, 2B2, Jupiter, FL, 33458, USA.
authors.forename: Hanumantharao
authors.initials: H
authors.lastname: Paritala
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, 2B2, Jupiter, FL, 33458, USA.
authors.forename: Prakash B
authors.initials: PB
authors.lastname: Palde
authors.affiliation: Department of Chemistry, The Scripps Research Institute, 130 Scripps Way, 2B2, Jupiter, FL, 33458, USA. kcarroll@scripps.edu.
authors.forename: Kate S
authors.initials: KS
authors.lastname: Carroll
mesh_terms: D000250:Adenosine Phosphosulfate / Q000737:chemistry / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006801:Humans; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013455:Sulfur / Q000378:metabolism*
chemical_list: D000250:Adenosine Phosphosulfate; D013455:Sulfur; D010088:Oxidoreductases
keywords: antibiotic target; directed evolution; enzymes; reduction; sulfate