Molecular cloning and mutational analysis of the ddsA gene encoding decaprenyl diphosphate synthase from Gluconobacter suboxydans.
Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquinone (Q)-10. We have cloned the synthase gene, designated ddsA, from Gluconobacter suboxydans and expressed it in Escherichia coli. Sequence analysis revealed the presence of an ORF of 948 bp capable of encoding a 33,898-Da polypeptide that displays high similarity (30-50%) to other prenyl diphosphate synthases. Expression of the ddsA gene complemented the lethality resulting from a defect in the octaprenyl diphosphate synthase gene of E. coli and produced Q-10, indicating that Q-10 can substitute for the function of Q-8. The His-tagged DdsA protein was purified to characterize its enzymatic properties. This enzyme required detergent (0.05% Triton X-100) and 10 mM Mg2+, for full activity. The Michaelis constants for geranyl diphosphate, all-E-farnesyl diphosphate and all-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 microM, respectively. Nine single-amino-acid substitutions were introduced upstream of conserved region II or VI. Most of the mutants showed a considerable decrease in catalytic activity or shortening of the ultimate chain length. However, the A70G mutant produced a longer-chain-length product than wild-type decaprenyl-PP synthase, and the A70Y mutant completely abolished the decaprenyl-PP synthase function, indicating that Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.
Okada
K
K
Department of Applied Bioscience and Biotechnology, Faculty of Life and Environmental Science, Nishikawatsu, Matsue, Japan.
Kainou
T
T
Tanaka
K
K
Nakagawa
T
T
Matsuda
H
H
Kawamukai
M
M
D016947:Acetobacteraceae / Q000201:enzymology / Q000235:genetics*; D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D015698:Genomic Library; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014451:Ubiquinone / Q000096:biosynthesis
D011994:Recombinant Proteins; D014451:Ubiquinone; D019883:Alkyl and Aryl Transferases; C039821:decaprenyl pyrophosphate synthetase; C025204:Ubiquinone Q2