br Introduction More than structurally different isoprenoids
Introduction More than 30,000 structurally different isoprenoids, such as steroids, carotenoids, prenyl side-chain of quinones, prenyl proteins, and natural rubber, exist in nature. In a biosynthetic pathway, linear prenyl diphosphates are common structures as a precursor of isoprenoids, as shown in Scheme 1, , , . Prenyltransferases can be classified into two major families, E- and Z-prenyl chain elongating enzymes. Furthermore, they salubrinal can be subdivided into three kinds of groups, short chain—(I), medium chain—(II), and long chain-elongating enzymes—(III), as shown in Table 1, , . E-farnesyl diphosphate synthase [EC 18.104.22.168] is included in a group of E-short chain elongating enzymes, which catalyze the condensation of isopentenyl diphosphate with dimethylallyl diphosphate or geranyl diphosphate (GPP) to give (all-E)-farnesyl diphosphate (FPP) as the ultimate product, as shown in Scheme 2. We have recently reported the reactivities of some allylic substrate homologs such as 8-hydroxygeranyl-, 8-methoxygeranyl-, 8-propoxygeranyl-, and 4-methoxymethoxydimethylallyl diphosphates by the use of wild and mutated types of Bacillus stearothermophilus farnesyl diphosphate synthases , . In this paper, we report substrate specificities of porcine liver FPP synthase and the wild and mutated type of B. stearothermophilus FPP synthases with respect to three kinds of allylic substrates, vinyldimethylallyl diphosphate (vinylDMAPP) and Z- (or E)-ethynyldimethylallyl diphosphate (Z- (or E)-ethynylDMAPP).
Results and discussion In order to investigate the reactivities of DMAPP homologs with vinyl or ethynyl group, we examined substrate specificities of thermostable FPP synthases of B. stearothermophilus and porcine liver FPP synthase as shown in Scheme 3 and Table 2.
Conclusion Scheme 3 illustrates the prenyl chain elongating reactions with DMAPP homologs having a vinyl or an ethynyl group. As the results, the reaction of 2 with 1 by FPP synthase of porcine liver gave 5a and 5b which stopped at the first stage and second stage of the sequential condensation of IPP, respectively. However, the similar reaction by the use of wild-type FPP synthase of B. stearothermophilus gave 5b, exclusively. 5b-OH or 6b-OH might be useful as synthons for the biologically active substance, such as juvenile hormone. On the other hand, the reaction of 3 with 1 by use of wild-type of FPP synthase of B. stearothermophilus gave 6b, solely. Moreover, a mutated FPP synthase (Y81D) reaction of 3 with 1 gave three kinds of products: 6a (2.2%), 6b (0.4%), and 6c (19.5%). Using wild-type FPP synthase of the thermostable bacteria, the reaction of 4 with 1 gave only 7b as double condensation product.
Isoprenoids are natural compounds synthesized from isopentenyl diphosphate (IPP) via allylic diphosphates with various numbers of carbons (). There are three genes coding for prenyl diphosphate synthases in the chromosome of : for farnesyl diphosphate (FPP) synthase (), for octaprenyl diphosphate synthase (), and () for undecaprenyl diphosphate synthase (, ). FPP synthase catalyzes the sequential condensation of IPP with dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) to form all--FPP, which is the substrate for both octaprenyl diphosphate synthase and undecaprenyl diphosphate synthase. Both () and () are essential for growth. However, the -null mutant is viable, although its growth rate is lower than that of the wild-type strain (). There are low enzyme activities in the cell of the mutant for the condensation of IPP with DMAPP. We studied this activity to find enzymes that substitute for FPP synthase in the mutant. The -null mutant SF7 () and the wild-type strain W3110 were cultured in 2×LB medium () at 37°C. The resulting cells were collected by centrifugation at 6000×g for 20 min at 4°C and suspended in 10 mM potassium phosphate buffer (pH 7.4) containing 1 mM 2-mercaptoethanol, 5 mM EDTA, 2 mM phenylmethanesulfonyl fluoride, and 10 mM octyl-β-thioglucopyranoside. The suspension was sonicated 10 times for 10 s, and centrifuged at 18,000×g for 40 min. The activity for the condensation of IPP with DMAPP in the supernatant of SF7 was 5% that of W3110 (). To identify the enzymes responsible for this residual activity in the mutant, the cell-free homogenate of SF7 was subjected to DEAE-Toyopearl column chromatography, and the prenyl diphosphate synthase activity of each fraction was determined (). Between the two large peaks of undecaprenyl diphosphate synthase (peak A) and octaprenyl diphosphate synthase (peak B), there was a small peak of enzyme activity (peak N: fractions 38–44). These fractions were pooled and applied to a DEAE-Toyopearl 650 M column again. Three peaks of enzyme activity (N1, N2, and N3) appeared in the second DEAE Toyopearl column chromatography ().