45(P-2) プレニル二リン酸合成酵素の機能変換の試み : 変異酵素の基質特異性(ポスター発表の部)

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Farnesyl diphosphate (FPP) synthase and geranylgeranyl diphosphate (GGPP) synthase catalyze the condensations of isopentenyl diphosphate(IPP) with allylic primer to give FPP and GGPP as a final product, respectively. These enzymes were cloned and these amino acid sequences have been determined. The comparison of amino acid sequence indicated the presence of seven conserved motifs. The second conserved motif include the sequence DDXXD which is holding the diphosphate group of allylic substrate. It has been reported that the fifth amino acid before this motif regulates the chain length of product. We constructed mutated FPP synthases (FPSs) from Bacillus stearothermophilus, in which tyrosine(Y)-81 was substituted with glycine (Y81G), serine (Y81S), arginine (Y81R) and aspartic acid (Y81D), and mutated GGPP synthases (GGPSs) from Sulfolobus acidocaldarius, in which phenylalanine (F)-77 was replaced with glycine (F77G) or serine (F77S). Y81 in FPS and F77 in GGPS are 5th amino acid before DDXXD, respectively. Then we studied the substrate specificities of these mutated enzymes using substrate analogs having oxygen atom in their prenyl chain (Fig.1). Interestingly the substrate specificities of the mutated FPSs (Y81G, Y81S, Y81R or Y81D) are dramatically altered. Y81G and Y81R accepted the analog 24 in 1.6 times higher than GPP (Fig.2). The analog 24 is hardly accepted by the wild FPS. The analog 20 was easily accepted by Y81S. On the other hand, the analog 3, which has only hydrocarbon group in its prenyl chain and is acceptedby the wild enzyme, can be hardly accepted by the mutanted enzyme. Furthermore, the mutated GGPSs (F77G and F77S), can also accept the oxygen containing analogs 21 and 22 which are hardly accepted by the wild GGPS (Fig.3). These results revealed that the single substitution of 5th amino acid of FPS or 5th amino acid of GGPS before DDXXD motifs with hydrophilic amino acids alter the substrate specificities of both enzymes, respectively. And these findings suggest that the mutanted enzymes have the wider possibility in the application to organic synthesis.
天然有機化合物討論会の論文
2001-09-01

45(P-2) プレニル二リン酸合成酵素の機能変換の試み : 変異酵素の基質特異性(ポスター発表の部)

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