1 中鎖プレニル二リン酸合成酵素の構造と反応機構の解析(口頭発表の部)

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Medium-chain (E)-prenyl diphosphate synthases are of special interest because of their heteromeric structures in contrast to other classes of prenyltransferases that are tightly coupled homodimers. Heptaprenyl diphosphate (HepPP) synthase of Bacillus subtilis is composed of two dissociable subunits, component I and component II, which are encoded by two cistrons in a novel gene cluster of gerC operon. This enzyme essentially requires the coexistence of both subunits for its catalysis. Expression vector systems for the two structural genes, gerC1 and gerC3, were constructed separately, and the two components were overproduced in Escherichia coli cells. After purification, their dynamic interactions in forming a catalytically active complex were investigated by gel filtration and immunoblotting analyses. When a mixture of the two components that had been preincubated in the presence of Mg^<2+> and farnesyl diphosphate (FPP) was subjected to Superdex 200 gel filtration, a significant elution peak appeared in a region earlier than those observed when they were chromatographed individually. This fraction contained both components I (GerC1) and -II (GerC3), and it corresponded to a molecular mass that is in accord with the sum of the values of the two components. Cross-linking studies indicate that the two essential subunits, FPP, and Mg^<2+> form a ternary complex, which seems to represent a catalytically active state of the HepPP synthase. On the other hand, no complex was formed in the presence of isopentenyl diphosphate or inorganic pyrophosphate and Mg^<2+>. A photoaffinity analogue of FPP was shown to preferentially label the GerC1 subunit, suggesting that GerC1 possesses a specific affinity for the allylic substrate. Furthermore, the photoaffinity labeling of GerC1 significantly increased in the presence of GerC3. The mechanism of catalysis of this unique heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of amphipathic product from soluble substrates.
天然有機化合物討論会の論文
1998-08-31

1 中鎖プレニル二リン酸合成酵素の構造と反応機構の解析(口頭発表の部)

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