The Carboxyl-terminal Region of the Geranylgeranyl Diphosphate Synthase is Indispensable for the Stabilization of the Region Involved in Substrate Binding and Catalysis

Abstract

Rat geranylgeranyl diphosphate synthase (GGPS) and its deletion mutants from the carboxyl terminus were analysed using Escherichia coli harbouring pACYC-crtIB, which contains crtI and crtB encoding the carotenoid biosynthetic enzymes. Mutants (delta-4, -8, -12 and -16) produced lycopene-derived red colour, but mutants (delta-17, -18, -19, -20, -23, -57 and -70) did not. The histidine-tagged mutants (delta-4, -8, -12 and -16) were overexpressed in E. coli BL21 (DE3) and purified in a stable form by nickel affinity chromatography except for one mutant (delta-16). The farnesyl-transferring activities of wild-type GGPS, delta-4, -8 and -12 mutants were relatively in a ratio of 1.0, 0.84, 0.26 and 0.0015. Each Km value of the four recombinants were estimated to be 0.71, 2.0 2.8 and 55 microM for farnesyl diphosphate and to be 2.9, 5.1, 56 and >100 microM for isopentenyl diphosphate, respectively. Allylic substrate specificities of these recombinants were estimated by quantitative analysis of the products, revealing that delta-8 and -12 mutants lack the ability to accept dimethylallyl and geranyl diphosphates compared to wild-type GGPS and delta-4 mutant. These results suggest that the KMFTEENE residing on the carboxyl-terminal sequence of GGPS stabilizes the active region involved in the substrate binding and catalysis.

GGPS1 Mutations Cause Muscular Dystrophy/Hearing Loss/Ovarian Insufficiency Syndrome

“…The pathogenic variants fell within a specific 5 amino acid region toward the C terminus around the start of helix 11, with as of yet unclear function (Fig 3A). In animals, geranylgeranyl diphosphate synthase is formed as a hexamer and in certain circumstances as an octamer, whereas in plants it is a dimer 17 . In silico modeling of GGPPS hexamer shows that this domain, in which all but one of the pathogenic variants (P15S) were clustered, is located toward the outside of the hexamer and not in the barrel where the catalytic core of the enzyme is located (Fig 4A).…”

“…In animals, geranylgeranyl diphosphate synthase is formed as a hexamer and in certain circumstances as an octamer, whereas in plants it is a dimer. 17 In silico modeling of GGPPS hexamer shows that this domain, in which all but one of the pathogenic variants (P15S) were clustered, is located toward the outside of the hexamer and not in the barrel where the catalytic core of the enzyme is located (Fig 4A). The first α-helix containing the P15S mutation is conserved and involved in the formation of the trimer from the dimers (see Fig 3A).…”