Dinner formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone

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.