Prenyl diphosphate synthetase genes

Details Prenyl diphosphate synthetase genes Prenyl diphosphate synthetase genes

1997-06-11

2001-01-16

Slobodyansky, Elizabeth (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Transferase other than ribonuclease

C435S069100, C435S183000, C435S252300, C435S320100, C536S023200, C536S023700

Reexamination Certificate

active

06174715

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for producing the peptide of a prenyl diphosphate synthetase, a method for producing an active type prenyl diphosphate synthetase, a DNA coding for the synthetase, a recombinant vector comprising the DNA and a transformant transformed with the vector.
2. Description of the Prior Art
An extremely wide variety of isoprenoid compounds are found in natural creatures from bacteria to higher eukaryotes. For example, steroids, carotenoids, polyprenols which are sugar carriers, quinones, tRNA modified with isopentenyladenine, prenylated proteins and the like may be enumerated. All of these isoprenoids are biosynthesized through prenyl diphosphate as an intermediate which is produced by a prenyl diphosphate synthetase (FIG.
1
).
The “prenyl diphosphate synthetase” is a general term for those enzymes which catalyze a reaction that condensation-polymerizes prenyl diphosphate (an allylic primer) and 3-isopentenyl diphosphate (IPP) to produce polyprenyl diphosphate.
Prenyl diphosphate synthetases are divided into two groups. One group consists of enzymes that catalyze a condensation reaction in which the double bond formed by each condensation of IPP is of E type. The other group consists of enzymes that catalyze a condensation reaction in which the double bond formed by each condensation of IPP is of Z type. Further, the maximum length of the isoprene chain which each prenyl diphosphate synthetase can produce is fixed. Since the hydrophobic property of a product varies depending of the isoprene chain length of the product, there is great difference in the mode of requirement for the activity of enzymes. When bacterial enzymes are compared in terms of the mode of requirement, prenyl diphosphate synthetases are classified into the following four groups.
(1) Prenyl diphosphate synthetase I (E type, short chain prenyl diphosphate synthetase)
(i) Geranyl diphosphate (GPP) synthetase (Sagami, H. et al., (1978) Biochem. Biophys. Res. Commun., 85, 575) (C
5
→C
10
)
The expression “C
5
→C
10
” means that the subject synthetase catalyzes the synthesis from a compound with 5 carbon atoms to a compound with 10 carbon atoms (hereinafter, this indication has a similar meaning.)
(ii) Farnesyl diphosphate (FPP) synthetase (Takahashi, I. and Ogura, K., (1981) J. Biochem. 89, 1581; Fujisaki, S. et al., (1986) J. Biochem., 99, 1327) (C
5
→C
10
)
(iii) Geranylgeranyl diphosphate (GGPP) synthetase (Takahashi, I. and Ogura, K., (1982) J. Biochem. 92, 1527; Sagami, H. and Ogura, K., (1981) J. Biochem., 89, 1573) (C
5
→C
20
)
(2) Prenyl diphosphate synthetase II (E type, medium chain prenyl diphosphate synthetase)
(i) Hexaprenyl diphosphate (HexPP) synthetase (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610) (C
15
→C
30
)
(ii) Heptaprenyl diphosphate (HepPP) synthetase (Takahashi, I. et al., (1980) J. Biochem., 255, 4539) (C
15
→C
35
)
(3) Prenyl diphosphate synthetase III (E type, long chain prenyl diphosphate synthetase)
(i) Octaprenyl diphosphate (OctPP) synthetase (Fujisaki, S. et al., (1986), J. Biochem., 99, 1327) (C.
15
→C
40
)
(ii) Nonaprenyl diphosphate (NonPP) synthetase (Sagami, H. et al., (1977) Biochemistry, 16, 4616) (C
10
→C
45
) Decaprenyl diphosphate (DecPP) synthetase (Ishii, K. et al., (1983) Biochem. Biophys. Res. Commun., 116, 500) (C
15
→C
50
)
(4) Prenyl diphosphate synthetase IV (Z type, long chain prenyl diphosphate synthetase)
(i) Z-nonaprenyl diphosphate synthetase (Ishii, K. et al., (1986) Biochem. J., 233, 773) (C
15
→C
45
)
(ii) Undecaprenyl diphosphate (UPP) synthetase (Takahashi, I. and Ogura, K. (1982) J. Biochem., 92, 1527; Keenman, M.V. and Allen, C.M. (1974) Arch. Biochem. Biophys., 161, 375)
(iii) Dehydrodolichyl diphosphate (deDolPP) synthetase (Sagami, H. et al., (1989) Biochem. Biophys. Acta. 1002, 218) (C
15
→C
85-105
)
Prenyl diphosphate synthetase I successively condensates 3-isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) generated by isomerization of IPP as an allylic primer to thereby synthesize a short chain, totally E type prenyl diphosphate with 20 or less carbon atoms. This product serves as a precursor for steroids, carotenoids or prenylated proteins. Further, geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) also serve as an allylic primer substrate for a medium- or long-chain prenyl diphosphate synthetase.
Hexaprenyl diphosphate synthetase (HexPS) and heptaprenyl diphosphate synthetase (HepPS) belong to prenyl diphosphate synthetase II. These enzymes synthesize hexaprenyl diphosphate and heptaprenyl diphosphate, respectively, without DMAPP nor GPP as a primer but using FPP as an allylic primer. The products are highly hydrophobic and serve as precursors for the side chains of menaquinones or ubiquinones in organisms having these enzymes. These prenylquinones play important roles in the respiratory chain or the electron transport system in photosynthesis.
Any member of prenyl diphosphate synthetase II is an enzyme composed of two essential proteins which do not have the catalytic activity independently. However, the enzyme has a property that in the presence of substrates for the enzyme, the two proteins associate with each other and exhibit the catalytic activity (Yoshida, I. et al., (1989) Biochem. Biophys. Res. Commun., 160, 448). In this point, the enzyme of this group is greatly different from other prenyl diphosphate synthetase.
As a microorganism producing prenyl diphosphate synthetase II,
Micrococcus luteus, Bacillus subtilis
and the like are known (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610; Takahashi, I. et al., (1980) J. Biol. Chem., 255, 4539). The following facts have been shown on the two components (designated “component A” and “component B”) of HexPS from
Micrococcus luteus
B-P 26 and on the two components (designated “component I” and “component II”) of HepPS of
Bacillus subtilis.
a) Component A and component I are relatively high in thermostability, whereas component B and component II have thermostability as low as that of other enzymes derived from mesophiles (Fujii, H. et al., (1982) J. Biol. Chem., 257, 14610).
b) There is no interchangeability between component A and component I. In other words, neither a combination of component A and component II nor a combination of component I and component B exhibits enzyme activity (Fujii, H. et al., (1983) FEBS Lett., 161, 257).
c) Component B and component II are affected by SH reagent and arginine-specific reagent to lower the enzyme activity remarkably, whereas component A and component I are not affected by these reagents (Yoshida, I. et al., (1989) Biochem. Biophys. Acta, 995, 138).
Octaprenyl diphosphate synthetase (OctPS), nonaprenyl diphosphate synthetase (NonPS) and the like belong to prenyl diphosphate synthetase III. Like prenyl diphosphate synthetase I, these enzymes are a homodimeric protein composed of identical subunits. They exhibit the catalytic activity by themselves. However, in order to maintain the turnover as a catalyst, they require a proteinaceous factor which removes hydrophobic products from their active site (Ohnuma, S. et al., (1991) J. Biol. Chem., 266, 23706). This activator is interchangeable and exhibits activating action against any enzyme belonging to prenyl diphosphate synthetase III (Ohnuma, S. et al., (1991) J. Biol. Chem., 266, 23706).
Enzymes belonging to prenyl diphosphate synthetase IV condensate IPP in the Z-structual form to synthesize polyprenyl diphosphate of E-and-Z mixed type using a short-chain prenyl diphosphate (GPP, FPP) as a primer substrate. Bacterial undecaprenyl diphosphate synthetase (UPS) and eukaryotic dehydrodolichyl diphosphate synthetase (deDolPS) are included in this group. A large number of these enzymes are a membrane-bound protein and when solubilized with a surfactant or the like, they require the addition of a surfactant such as Triton X-100 for the manifestation of their activity in almost all cases (

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