Kenzo Funatsuki

acrB Mutation Located at Carboxyl-terminal Region of Gyrase B Subunit Reduces DNA Binding of DNA Gyrase

Mutations that exhibit susceptibility to acriflavine have been isolated and classified as acrmutations in Escherichia coli. We cloned theacrB gene, which has been identified as a mutation of thegyrB gene, and found a double point mutation altering two consecutive amino acids (S759R/R760C) in the COOH-terminal region of the gyrase B subunit. The mutant B subunit was found to associate with the A subunit to make the quaternary structure, and the reconstituted gyrase showed an 80-fold reduction of specific activity in DNA supercoiling assay; the sensitivity to acriflavine was not different in the same unit of wild-type and mutant gyrases. The mutant enzyme retained intrinsic ATPase activity, but DNA-dependent stimulation was observed infrequently. A gel shift assay showed that acriflavine inhibited the DNA binding of gyrase. The acrBmutation also reduced significantly the DNA binding of gyrase but did not change the sensitivity to acriflavine. These results revealed that the acrB mutation is related to the inhibitory mechanism of acriflavine; and the acriflavine sensitivity of the mutant, at leastin vitro, is caused mainly by reduction of the enzyme activity. Further, our findings suggest that the COOH-terminal region of the B subunit is essential for the initial binding of gyrase to the substrate DNA.

Biosynthesis of kelsoene in cultured cells of liverworts Ptychanthus striatus

The most uncommon tricyclic sesquiterpenes, kelsoene 1 and prespatane 2, were isolated from cultured cells of liverwort Ptychanthus striatus, and the labelling patternof kelsoene biosynthesized from exogenous [2-13C]-mevalonate suggested that kelsoene is biosynthesized from germacradienyl cation via alloaromadendranyl cation.