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 pre...

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Bibliographic Details
Published in:Insect biochemistry and molecular biology 2023-10, Vol.161, p.104001-104001, Article 104001
Main Authors: Ducker, Charles, French, Stanley, Pathak, Monika, Taylor, Harry, Sainter, Adam, Askem, William, Dreveny, Ingrid, Santana, Antônio Euzébio Goulart, Pickett, John A., Oldham, Neil J.
Format: Article
Language:English
Subjects:
aggregation pheromones
diterpenoids
divalent metals
geranylgeranyl diphosphate synthase
humans
insect biochemistry
kinetics
leishmaniasis
Lutzomyia longipalpis
molecular biology
molecular weight
tropical diseases
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Summary: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. [Display omitted] •Geranylgeranyl diphosphate synthase (lzGGPPS) from Lutzomyia longipalpis expressed and functionally tested.•Mg2+ identified as the most efficient cofactor of lzGGPPS.•lzGGPPS displayed product inhibition by GGPP.•Assessment of native molecular weight in solution suggests the enzyme is hexameric.
ISSN:0965-1748
1879-0240
DOI:10.1016/j.ibmb.2023.104001