Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis

Ducker, Charles; French, Stanley; Pathak, Monika; Taylor, Harry; Sainter, Adam; Askem, William; Dreveny, Ingrid; Goulart Santana, Antônio Euzébio; Pickett, John A.; Oldham, Neil J.

Abstract

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.

Citation

Ducker, C., French, S., Pathak, M., Taylor, H., Sainter, A., Askem, W., Dreveny, I., Goulart Santana, A. E., Pickett, J. A., & Oldham, N. J. (2023). Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis. Insect Biochemistry and Molecular Biology, 161, Article 104001. https://doi.org/10.1016/j.ibmb.2023.104001

• Journal Article Type: Article
• Acceptance Date: Aug 20, 2023
• Online Publication Date: Aug 22, 2023
• Publication Date: 2023-10
• Deposit Date: Aug 29, 2023
• Publicly Available Date: Aug 30, 2023
• Journal: Insect Biochemistry and Molecular Biology
• Print ISSN: 0965-1748
• Electronic ISSN: 1879-0240
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 161
• Article Number: 104001
• DOI: https://doi.org/10.1016/j.ibmb.2023.104001
• Keywords: Insect Science, Molecular Biology, Biochemistry
• Public URL: https://nottingham-repository.worktribe.com/output/24585981
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0965174823000954?via%3Dihub