MOLECULAR-CLONING AND MUTATIONAL ANALYSIS OF THE DDSA GENE ENCODING DECAPRENYL DIPHOSPHATE SYNTHASE FROM GLUCONOBACTER SUBOXYDANS

Decaprenyl diphosphate (decaprenyl-PP) synthase catalyzes the consecut ive condensation of isopentenyl diphosphate with allylic diphosphates to produce decaprenyl-PP, which is used for the side chain of ubiquino ne (Q)-10. We have cloned the synthase gene, designated ddsA, from Glu conobacter suboxy-dans 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 oth er prenyl diphosphate synthases. Expression of the ddsA gene complemen ted the lethality resulting from a defect in the octaprenyl diphosphat e 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 p urified 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 diphospha te and a Il-E-geranylgeranyl diphosphate were 7.00, 0.50 and 0.32 mu M , respectively. Nine single-amino-acid substitutions were introduced u pstream of conserved region II or VI. Most of the mutants showed a con siderable 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 co mpletely abolished the decaprenyl-PP synthase function, indicating tha t Ala70 is important for enzyme activity and the determination of the chain-length properties of DdsA.