Abstract
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-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants 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 produced Q-5 and Q-6, indicating that these residues are involved in the determination 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 harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length.
Highlights
Ubiquinone (Q)1 is an essential component of the electron transport system associated with aerobic growth and oxidative phosphorylation in many organisms
Complementation of E. coli ispB- and S. cerevisiae COQ1defective Mutants with Mutant ispB Genes—E. coli DH10B was transformed with the plasmids containing mutant ispB genes, and the transformants were used in ubiquinone analysis
To detect the actual ubiquinone species produced by the product of the mutant ispB gene, E. coli KO229/pKA3, which is defective for the genomic ispB gene, but retains ispB in a plasmid, was transformed with the plasmids containing the mutant ispB genes
Summary
Q, ubiquinone; GGPP, geranylgeranyl diphosphate; FPP, farnesyl diphosphate; IPP, isopentenyl diphosphate; PCR, polymerase chain reaction; GST, glutathione S-transferase; NTA, nitrilotriacetic acid; HPLC,high pressure liquid chromatography. When COQ2, which encodes 4-hydroxybenzoate-hexaprenyldiphosphate transferase in S. cerevisiae, was expressed in an E. coli ubiA mutant cell line, the transformant produce Q-8, but not Q-6 [10] These results indicate that prenyl-diphosphate synthase determines the chain length of ubiquinone and that 4-hydroxybenzoate-polyprenyltransferases can accept the various isoprenoid chains as a substrate. Long-chain polyprenyl-diphosphate synthases (C40, C45, and C50) catalyze the condensation of FPP, which acts as a primer, and IPP to produce each prenyl diphosphate with various chain lengths. These enzymes possess seven conserved regions including two DDXXD motifs that are binding sites for the substrates in association with Mg2ϩ [13, 14]. We found that IspB forms a homodimer that is important for the determination of isoprenoid chain length
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