2 SUBUNITS OF HEPTAPRENYL DIPHOSPHATE SYNTHASE OF BACILLUS-SUBTILIS FORM A CATALYTICALLY ACTIVE COMPLEX

Heptaprenyl diphosphate synthase of Bacillus subtilis, which participa tes in the biosynthesis of the side chain of menaquinone-7, is compose d of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon [Zhang , Y.-W., et al. (1997) J. Bacteriol. 179, 1417-1419]. This enzyme esse ntially requires the coexistence of both subunits for its catalysis. E xpression vector systems for the two structural genes, gerC1 and gerC3 , were constructed separately, and the two components were overproduce d in Escherichia coli cells. After purification, their dynamic interac tions in forming a catalytically active complex were investigated by g el filtration and immunoblotting analyses. When a mixture of the two c omponents that had been preincubated in the presence of Mg2+ and farne syl diphosphate was subjected to Superdex 200 gel filtration, a signif icant elution peak appeared in a region earlier than those observed wh en they were chromatographed individually. This fraction contained bot h components I and II, and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-lin king studies indicate that the two essential subunits, farnesyl diphos phate, and Mg2+ form a ternary complex which seems to represent a cata lytically active state of the heptaprenyl diphosphate synthase, On the other hand, no complex was formed in the presence of isopentenyl diph osphate or inorganic pyrophosphate and Mg2+. A photoaffinity analogue of farnesyl diphosphate was shown to preferentially label the componen t I protein, suggesting that component I possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of component I significantly increased in the presence of component II. The mechanism of catalysis of this unique heteromeric enzyme is unders tood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic product from soluble substrates.