T. Kainou et al., Dinner formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone, J BIOL CHEM, 276(11), 2001, pp. 7876-7883
Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is
a key component of the respiratory chain and is an important antioxidant.
In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-dip
hosphate synthase, which is encoded by an essential gene, ispB. To determin
e how IspB regulates the length of the isoprenoid, we constructed 15 ispB m
utants and expressed them in E. coli and Saccharomyces cerevisiae. The Y38A
and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant p
roduced Q-5 and Q-6, indicating that these residues are involved in the det
ermination of chain length. E. coli cells (ispB::cat) harboring an Arg-321
mutant were temperature-sensitive for growth, which indicates that Arg-321
is important for thermostability of IspB. Intriguingly, E. coli cells harbo
ring wild-type ispB and the A79Y mutant produced mainly Q-6, although the a
ctivity of the enzyme with the A79Y mutation was completely abolished. When
a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-t
agged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoi
d. These results indicate that although the A79Y mutant is functionally ina
ctive, it can regulate activity upon forming a heterodimer with wild-type I
spB, and this dimer formation is important for the determination of the iso
prenoid chain length.