Control Activity of Yeast Geranylgeranyl Diphosphate
Synthase from Dimer Interface through H‑Bonds and Hydrophobic
Interaction
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Abstract
Previously we showed that
yeast geranylgeranyl diphosphate synthase (GGPPS) becomes an inactive
monomer when the first N-terminal helix involved in dimerization is
deleted. This raises questions regarding why dimerization is required
for GGPPS activity and which amino acids in the dimer interface are
essential for dimerization-mediated activity. According to the GGPPS
crystal structure, three amino acids (N101, N104, and Y105) located
in the helix F of one subunit are near the active site of the other
subunit. As presented here, when these residues were replaced individually
with Ala caused insignificant activity changes, N101A/Y105A and N101A/N104A
but not N104A/Y105A showed remarkably decreased <i>k</i><sub>cat</sub> values (200–250-fold). The triple mutant N101A/N104A/Y105A
displayed no detectable activity, although dimer was retained in these
mutants. Because N101 and Y105 form H-bonds with H139 and R140 in
the other subunit, respectively, we generated H139A/R140A double mutant
and found it was inactive and became monomeric. Therefore, the multiple
mutations apparently influence the integrity of the catalytic site
due to the missing H-bonding network. Moreover, Met111, also on the
highly conserved helix F, was necessary for dimer formation and enzyme
activity. When Met111 was replaced with Glu, the negative-charged
repulsion converted half of the dimer into a monomer. In conclusion,
the H-bonds mainly through N101 for maintaining substrate binding
stability and the hydrophobic interaction of M111 in dimer interface
are essential for activity of yeast GGPPS