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Figshare Pub Date : 2017-11-21 DOI: 10.1021/jacs.7b09384.s001

Miha Purg (1316844), Mikael Elias (128712), Shina Caroline Lynn Kamerlin (686551)

{"title":"SimilarnActive Sites and Mechanisms Do Not Lead to Cross-Promiscuity in OrganophosphatenHydrolysis: Implications for Biotherapeutic Engineering","authors":"Miha Purg (1316844), Mikael Elias (128712), Shina Caroline Lynn Kamerlin (686551)","doi":"10.1021/jacs.7b09384.s001","DOIUrl":"https://doi.org/10.1021/jacs.7b09384.s001","url":null,"abstract":"Organophosphate hydrolases are proficientncatalysts of the breakdown of neurotoxic organophosphates and havengreat potential as both biotherapeutics for treating acute organophosphatentoxicity and as bioremediation agents. However, proficient organophosphatasesnsuch as serum paraoxonase 1 (PON1) and the organophosphate-hydrolyzingnlactonase <i>Sso</i>Pox are unable to hydrolyze bulkyorganophosphatesnwith challenging leaving groups such as diisopropyl fluorophosphaten(DFP) or venomous agent X, creating a major challenge for enzyme design.nCuriously, despite their mutually exclusive substrate specificities,nPON1 and diisopropyl fluorophosphatase (DFPase) have essentially identicalnactive sites and tertiary structures. In the present work, we usenempirical valence bond simulations to probe the catalytic mechanismnof DFPase as well as temperature, pH, and mutational effects, demonstratingnthat DFPase and PON1 also likely utilize identical catalytic mechanismsnto hydrolyze their respective substrates. However, detailed examinationnof both static structures and dynamical simulations demonstrates subtlenbut significant differences in the electrostatic properties and solventnpenetration of the two active sites and, most critically, the rolenof residues that make no direct contact with either substrate in actingnas “specificity switches” between the two enzymes. Specifically,nwe demonstrate that key residues that are structurally and functionallyncritical for the paraoxonase activity of PON1 prevent it from beingnable to hydrolyze DFP with its fluoride leaving group. These insightsnexpand our understanding of the drivers of the evolution of divergentnsubstrate specificity in enzymes with identical active sites and guidenthe future design of organophosphate hydrolases that hydrolyze compoundsnwith challenging leaving groups.","PeriodicalId":521663,"journal":{"name":"Figshare","volume":"1 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2017-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147883429","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

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