Possible Mechanisms Modulating Disruption of S. cerevisiae Geranylgeranyl Pyrophosphate Synthase Dimer into Monomer and Hexamerization Study of Human Geranylgeranyl Pyrophosphate Synthase
Date Issued
2008
Date
2008
Author(s)
Chang, Ying-Hsuan
Abstract
Geranylgeranyl pyrophosphate synthase (GGPPs) catalyzes a condensation reaction of farnesyl pyrophosphate (FPP) with isopentenyl pyrophosphate (IPP) to generate C20 geranylgeranyl pyrophosphate (GGPP), which is a precursor for carotenoids, chlorophylls, geranylgeranylated proteins, and archaeal ether linked lipids. Distinct from other known structures of trans-prenyltransferases, the N-terminal 17 amino acids (9-amino acid helix A and the following loop) of this GGPPs protrude from the helix core into the other subunit and contribute to the tight dimer formation. In previous study, we have known that L8 and I9 were essential in dimerization (Lo, C. H. et al., unpublished data). The double mutant L8G/I9G can completely disrupt a dimeric S. cerevisiae GGPPs into a monomer with at least 103-fold reduction in activity (Lo, C. H. et al., unpublished data). In order to investigate mechanisms by which L8G/I9G disrupt the dimer, I observed the X-ray crystal structure of the enzyme and proposed that two sets of interactions networking with L8 and I9 may stabilize the dimerization (Figure 4 and Figure 5). One mechanism is that L8 and I9 make vdW contacts with L163 and M167, whose neighboring residues, N168, K169, and G171, are within vdW contact of G141, N137 and L138 located at the main dimer interface (Figure 4). In particular, K169 forms a salt-bridge with D145, which in turn forms a backbone-backbone hydrogen bond with G141, which itself is hydrogen bonded to N137, both of which are at the main dimer interface (Figure 4). H139, close to L138 and G141, forms a hydrogen bond with N101 in the other chain (Figure 4). The other is that L8 and I9 make vdW contacts with L200 and I203 whose neighboring residue, N199, is hydrogen bonded to R175, which in turn forms a salt-bridge with E134 located near the main dimer interface (Figure 5). This interaction is reinforced since R175 and E134 are also both hydrogen bonds to R179 (Figure 5). I then performed site-directed mutagenesis studies and used analytic ultracentrifuge (AUC) to characterize the mutants. Single mutant of L163G, M167G, D145K, or N101G involved in one possible mechanism and L200G, I203G, E134A, or R175A involved in the other did not result in the disruption of dimer to monomer, whereas double mutant of M167G/N199A involved in both possible mechanisms resulted in a mixture of dimer and monomer. This indicated that both possible mechanisms were involved in modulating disruption of S. cerevisiae geranylgeranyl pyrophosphate synthase dimer into monomer.n human geranylgeranyl pyrophosphate synthase, there is no N-terminal arm protruding into the other subunit, whereas it forms a stable hexamer. The 3-D structure of human GGPPs suggested that the first helix at N-terminus may play an important role in inter-dimer interaction to stabilize the structure of hexamer. To confirm this, the human GGPPS without the first 21 amino acids were characterized using analytic ultracentrifugation (AUC).This resulted in a mixture of monomer and dimer. To identify the critical amino acids in the N-terminal helix for hexamerization, I observed the X-ray crystal structure of the enzyme and assumed E14, Y18, or Q21 may involved in inter-dimer interactions by binding with Y246 and T228 to stabilize the hexamer. To confirm this, I generated E14G/Y18G/Q21G, E14G, Y18G, and Q21G and characterized them respectively using AUC. The triple mutant E14G/Y18G/Q21G became a mixture of monomer and tetramer, whereas the single mutant E14G, Y18G, and Q21G remained as a hexamer. The results suggested that E14, Y18, and Q21 together provided critical roles for the hGGPPs inter-dimer interactions and stabilize the structure of hexamer.
Subjects
Geranylgeranyl Pyrophosphate Synthase
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