Timothy J Grunkemeyer, David G Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery
{"title":"对氧杂环戊烯酶-1 机制的启示:比较六叶片 β-螺旋桨水解酶的反应性。","authors":"Timothy J Grunkemeyer, David G Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery","doi":"10.1021/acs.biochem.8b01115","DOIUrl":null,"url":null,"abstract":"<p><p>The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase, bacterial drug responsive protein 35, and mammalian senescence marker protein 30. We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites and the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca<sup>2+</sup>-coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.</p>","PeriodicalId":28,"journal":{"name":"Biochemistry Biochemistry","volume":" ","pages":"3287-3299"},"PeriodicalIF":2.9000,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Insights into the Mechanism of Paraoxonase-1: Comparing the Reactivity of the Six-Bladed β-Propeller Hydrolases.\",\"authors\":\"Timothy J Grunkemeyer, David G Mata, Kiran Doddapaneni, Srividya Murali, Thomas J Magliery\",\"doi\":\"10.1021/acs.biochem.8b01115\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase, bacterial drug responsive protein 35, and mammalian senescence marker protein 30. We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites and the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca<sup>2+</sup>-coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.</p>\",\"PeriodicalId\":28,\"journal\":{\"name\":\"Biochemistry Biochemistry\",\"volume\":\" \",\"pages\":\"3287-3299\"},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2024-12-17\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Biochemistry Biochemistry\",\"FirstCategoryId\":\"1\",\"ListUrlMain\":\"https://doi.org/10.1021/acs.biochem.8b01115\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biochemistry Biochemistry","FirstCategoryId":"1","ListUrlMain":"https://doi.org/10.1021/acs.biochem.8b01115","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Insights into the Mechanism of Paraoxonase-1: Comparing the Reactivity of the Six-Bladed β-Propeller Hydrolases.
The mammalian protein paraoxonase-1 (PON1) has been explored as a promising bioscavenger treatment for organophosphorus (OP) agent poisoning, but it is not active enough to protect against many agents. Engineering is limited because PON1's catalytic mechanism is poorly understood; moreover, its native activity and substrate are unknown. PON1 is a calcium-bound six-bladed β-propeller hydrolase that shares high structural homology, a conserved metal-coordinating active site, and substrate specificity overlap with other members of a superfamily that includes squid diisopropylfluorophosphatase, bacterial drug responsive protein 35, and mammalian senescence marker protein 30. We hypothesized that, by examining the reactivity of all four hydrolases using a common set of conservative mutations, we could gain further insight into the catalytic mechanism of PON1. We chose a set of mutations to examine conserved Asp and Glu residues in the hydrolase active sites and the ligation sphere around the catalytic calcium and a His-His dyad seen in PON1. The wild-type (WT) and mutant hydrolases were assayed against a set of lactones, aryl esters, and OPs that PON1 is known to hydrolyze. Surprisingly, some mutations of Ca2+-coordinating residues, previously thought to be essential for turnover, resulted in significant activity toward all substrate classes examined. Additionally, merely maintaining WT-like charge in the active site of PON1 was insufficient for high activity. Finally, the H115-H134 dyad does not appear to be essential for catalysis against any substrate. Therefore, previously proposed mechanisms must be re-evaluated.
期刊介绍:
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