Jacob Kronenberg, Dustin Britton, Leif Halvorsen, Stanley Chu, Maria Jinu Kulapurathazhe, Jason Chen, Ashwitha Lakshmi, P Douglas Renfrew, Richard Bonneau, Jin Kim Montclare
{"title":"增压磷酸酯酶可提高副氧自由基的活性","authors":"Jacob Kronenberg, Dustin Britton, Leif Halvorsen, Stanley Chu, Maria Jinu Kulapurathazhe, Jason Chen, Ashwitha Lakshmi, P Douglas Renfrew, Richard Bonneau, Jin Kim Montclare","doi":"10.1093/protein/gzae015","DOIUrl":null,"url":null,"abstract":"Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge–charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with −14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermostability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C – 55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.","PeriodicalId":20681,"journal":{"name":"Protein Engineering, Design and Selection","volume":"18 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Supercharged Phosphotriesterase for improved Paraoxon activity\",\"authors\":\"Jacob Kronenberg, Dustin Britton, Leif Halvorsen, Stanley Chu, Maria Jinu Kulapurathazhe, Jason Chen, Ashwitha Lakshmi, P Douglas Renfrew, Richard Bonneau, Jin Kim Montclare\",\"doi\":\"10.1093/protein/gzae015\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge–charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with −14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermostability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C – 55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.\",\"PeriodicalId\":20681,\"journal\":{\"name\":\"Protein Engineering, Design and Selection\",\"volume\":\"18 1\",\"pages\":\"\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-09-18\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein Engineering, Design and Selection\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1093/protein/gzae015\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein Engineering, Design and Selection","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/protein/gzae015","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Supercharged Phosphotriesterase for improved Paraoxon activity
Phosphotriesterases (PTEs) represent a class of enzymes capable of efficient neutralization of organophosphates (OPs), a dangerous class of neurotoxic chemicals. PTEs suffer from low catalytic activity, particularly at higher temperatures, due to low thermostability and low solubility. Supercharging, a protein engineering approach via selective mutation of surface residues to charged residues, has been successfully employed to generate proteins with increased solubility and thermostability by promoting charge–charge repulsion between proteins. We set out to overcome the challenges in improving PTE activity against OPs by employing a computational protein supercharging algorithm in Rosetta. Here, we discover two supercharged PTE variants, one negatively supercharged (with −14 net charge) and one positively supercharged (with +12 net charge) and characterize them for their thermostability and catalytic activity. We find that positively supercharged PTE possesses slight but significant losses in thermostability, which correlates to losses in catalytic efficiency at all temperatures, whereas negatively supercharged PTE possesses increased catalytic activity across 25°C – 55°C while offering similar thermostability characteristic to the parent PTE. The impact of supercharging on catalytic efficiency will inform the design of shelf-stable PTE and criteria for enzyme engineering.