Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis

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

Characterisation of geranylgeranyl diphosphate synthase from the sandfly Lutzomyia longipalpis

Insect Biochem Mol Biol. 2023 Oct:161:104001. doi: 10.1016/j.ibmb.2023.104001. Epub 2023 Aug 22.

Affiliations

¹ School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
² School of Pharmacy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
³ Center of Engineering and Agrarian Science, Federal University of Alagoas, Maceio, Brazil.
⁴ School of Chemistry, Cardiff University, Main Building, Park Pl, Cardiff, CF10 3AT, UK.
⁵ School of Chemistry, University of Nottingham, University Park, Nottingham, NG7 2RD, UK. Electronic address: neil.oldham@nottingham.ac.uk.

PMID: 37619821
DOI: 10.1016/j.ibmb.2023.104001

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.