Charles Ducker , Stanley French , Monika Pathak , Harry Taylor , Adam Sainter , William Askem , Ingrid Dreveny , Antônio Euzébio Goulart Santana , John A. Pickett , Neil J. Oldham
{"title":"白蛉香叶基二磷酸合成酶的鉴定","authors":"Charles Ducker , Stanley French , Monika Pathak , Harry Taylor , Adam Sainter , William Askem , Ingrid Dreveny , Antônio Euzébio Goulart Santana , John A. Pickett , Neil J. Oldham","doi":"10.1016/j.ibmb.2023.104001","DOIUrl":null,"url":null,"abstract":"<div><p>Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the <em>Leishmania</em>-harbouring sandfly, <em>Lutzomyia longipalpis</em>, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (<em>lz</em>GGPPS) from <em>L. longipalpis</em>, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. <em>In vitro</em> enzymatic assays using LC-MS showed that <em>lz</em>GGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (<em>E</em>)-geranyl diphosphate (GPP), (<em>E,E</em>)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (<em>E,E,E</em>)-GGPP, while (<em>Z,E</em>)-FPP was also accepted with low efficacy. Comparison of metal cofactors for <em>lz</em>GGPPS highlighted Mg<sup>2+</sup> as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of <em>lz</em>GGPPS activity. The molecular weight in solution of <em>lz</em>GGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.</p></div>","PeriodicalId":330,"journal":{"name":"Insect Biochemistry and Molecular Biology","volume":null,"pages":null},"PeriodicalIF":3.2000,"publicationDate":"2023-08-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis\",\"authors\":\"Charles Ducker , Stanley French , Monika Pathak , Harry Taylor , Adam Sainter , William Askem , Ingrid Dreveny , Antônio Euzébio Goulart Santana , John A. Pickett , Neil J. Oldham\",\"doi\":\"10.1016/j.ibmb.2023.104001\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<div><p>Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the <em>Leishmania</em>-harbouring sandfly, <em>Lutzomyia longipalpis</em>, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (<em>lz</em>GGPPS) from <em>L. longipalpis</em>, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. <em>In vitro</em> enzymatic assays using LC-MS showed that <em>lz</em>GGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (<em>E</em>)-geranyl diphosphate (GPP), (<em>E,E</em>)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (<em>E,E,E</em>)-GGPP, while (<em>Z,E</em>)-FPP was also accepted with low efficacy. Comparison of metal cofactors for <em>lz</em>GGPPS highlighted Mg<sup>2+</sup> as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of <em>lz</em>GGPPS activity. The molecular weight in solution of <em>lz</em>GGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.</p></div>\",\"PeriodicalId\":330,\"journal\":{\"name\":\"Insect Biochemistry and Molecular Biology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":3.2000,\"publicationDate\":\"2023-08-22\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Insect Biochemistry and Molecular Biology\",\"FirstCategoryId\":\"97\",\"ListUrlMain\":\"https://www.sciencedirect.com/science/article/pii/S0965174823000954\",\"RegionNum\":2,\"RegionCategory\":\"农林科学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q2\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Insect Biochemistry and Molecular Biology","FirstCategoryId":"97","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0965174823000954","RegionNum":2,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis
Leishmaniasis is a debilitating and often fatal neglected tropical disease. Males from sub-populations of the Leishmania-harbouring sandfly, Lutzomyia longipalpis, produce the diterpene sex and aggregation pheromone, sobralene, for which geranylgeranyl diphosphate (GGPP) is the likely isoprenoid precursor. We have identified a GGPP synthase (lzGGPPS) from L. longipalpis, which was recombinantly expressed in bacteria and purified for functional and kinetic analysis. In vitro enzymatic assays using LC-MS showed that lzGGPPS is an active enzyme, capable of converting substrates dimethylallyl diphosphate (DMAPP), (E)-geranyl diphosphate (GPP), (E,E)-farnesyl diphosphate (FPP) with co-substrate isopentenyl diphosphate (IPP) into (E,E,E)-GGPP, while (Z,E)-FPP was also accepted with low efficacy. Comparison of metal cofactors for lzGGPPS highlighted Mg2+ as most efficient, giving increased GGPP output when compared against other divalent metal ions tested. In line with previously characterised GGPPS enzymes, GGPP acted as an inhibitor of lzGGPPS activity. The molecular weight in solution of lzGGPPS was determined to be ∼221 kDa by analytical SEC, suggesting a hexameric assembly, as seen in the human enzyme, and representing the first assessment of GGPPS quaternary structure in insects.
期刊介绍:
This international journal publishes original contributions and mini-reviews in the fields of insect biochemistry and insect molecular biology. Main areas of interest are neurochemistry, hormone and pheromone biochemistry, enzymes and metabolism, hormone action and gene regulation, gene characterization and structure, pharmacology, immunology and cell and tissue culture. Papers on the biochemistry and molecular biology of other groups of arthropods are published if of general interest to the readership. Technique papers will be considered for publication if they significantly advance the field of insect biochemistry and molecular biology in the opinion of the Editors and Editorial Board.