Nucleic acid molecules encoding isopentenyl monophosphate...

Details Nucleic acid molecules encoding isopentenyl monophosphate... Nucleic acid molecules encoding isopentenyl monophosphate...

1999-11-04

2001-05-22

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

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

Transferase other than ribonuclease

C435S183000, C435S252300, C435S320100, C435S419000, C514S04400A, C536S023600

Reexamination Certificate

active

06235514

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to nucleic acid sequences encoding isopentenyl monophosphate kinase, in particular to nucleic acid sequences encoding isopentenyl monophosphate kinase from peppermint.
BACKGROUND OF THE INVENTION
Isopentenyl diphosphate (IPP) is the central intermediate in the biosynthesis of isoprenoids in all organisms. In higher plants, the formation of IPP is compartmentalized. The mevalonate (MVA) pathway, the enzymes of which are localized to the cytosolic compartment, produces the precursor of triterpenes (sterols) and certain sesquiterpenes (Newman, J. D. & Chappell, J.,
Crit. Rev. Biochem. Mol. Biol.,
34:95-106 [1999]). In plastids, the deoxyxylulose-5-phosphate (DXP) pathway operates to supply IPP for the synthesis of monoterpenes and diterpenes (Eisenreich, W. et al.,
Tetrahedron Lett.,
38:3889-3892 [1997]; Eisenreich, W. et al.,
Proc. Natl. Acad. Sci. USA,
93:6431-6436 [1996]), several sequiterpenes (McCaskill, D. & Croteau, R.,
Planta,
197:49-56 [1995]), tetraterpenes (carotenoids), and the prenyl side-chains of chlorophyll and plastoquinone (Lichtenthaler, H. K. et al.,
FEBS Lett.,
400:271-274 [1997]).
In addition, there are examples of cooperation between the cytosolic and plastidial pathways in the biosynthesis of stress-induced and constitutively emitted volatile terpenoids from a variety of plants (Piel, J. et al.,
Angew. Chem. Int. Ed.,
37:2478-2481 [1998]), and constitutive sesquiterpenes of chamomile (Adam, K.-P. & Zapp, J.,
Phytochemistry,
48:953-959 [1998]). In mammals, where the DXP pathway is not known to operate, and in plants, the individual biosynthetic steps of the MVA pathway have been well-characterized (Goldstein, J. L. & Brown, M. S.,
Nature
(
London
), 343:425-430 [1990]; Bach, T. J.,
Crit. Rev. Biochem. Mol. Biol.,
34:107-122 [1999]). However, for the recently discovered DXP pathway, which also occurs in many eubacteria (Rohmer, M.,
Prog. Drug Res.,
50:135-154 [1998]), the biosynthetic sequence leading to the formation of IPP is still incompletely defined (The FIGURE).
The initial step of the pathway involves a condensation of pyruvate (C2 and C3) with D-glyceraldehyde-3-phosphate (GAP) to yield 1-deoxy-D-xylulose-5-phosphate (Rohmer, M.,
Biochem. J.,
295:517-524 [1993]; Broers, S. T. J., Ph.D. thesis, Eidgenössische Technische Hochschule, Zürich, Switzerland [1994]; Schwarz, M. K., Ph.D. thesis, Eidgenössische Technische Hochschule, Zürich, Switzerland [1994]; Rohmer, M. et al.,
J. Am. Chem. Soc.,
118:2564-2566 [1996]). The enzyme which catalyzes this reaction belongs to a novel family of transketolases, and the corresponding gene has been isolated from
Escherichia coli
(Sprenger, G. A. et al.,
Proc. Natl. Acad. Sci. USA,
94:12857-12862 [1997]; Lois, L. M. et al.,
Proc. Natl. Acad. Sci. USA,
95:2105-2110 [1997]), peppermint (Lange, B. M. et al.,
Proc. Natl. Acad. Sci. USA,
95:2100-2104 [1998]) and pepper (Bouvier, F. et al.,
Plant Physiol.,
117:1423-1431 [1998]). In the second step of this pathway, rearrangement and reduction of DXP yield 2-C-methyl-D-erythritol (MEP) (Duvold, T. et al.,
Tetrahedron Lett.,
38:4769-4772 [1997]; Duvold, T. et al.,
Tetrahedron Lett.,
38:6181-6184 [1997]; Sagner, S. et al.,
Tetrahedron Lett.,
39:2091-2094 [1998]) (The FIGURE). Recently, genes encoding this DXP reductoisomerase (DXR) have been cloned from
E. coli
(Takahashi, S. et al.,
Proc. Natl. Acad. Sci. USA,
95:9879-9884 [1998]), peppermint (Lange, B. M. & Croteau R.,
Arch. Biochem. Biophys.,
365:170-174 [1999]), and
Arabidopsis thaliana
(Lange, B. M. & Croteau R.,
Arch. Biochem. Biophys.,
365:170-174 [1999]; Schwender, J. et al.,
FEBS Lett.,
455:140-144 [1999]). To date, no other intermediates on the route to IPP, the terminal product of the DXP pathway (McCaskill, D. & Croteau R.,
Tetrahedron Lett.,
40:653-656 [1999]; Arigoni, D. et al.,
Proc. Natl. Acad. Sci. USA,
96:1309-1314 [1999]), have been identified.
As disclosed herein, sequencing of 1300 anonymous clones (expressed sequence tags, ESTs) from a cDNA library constructed from mRNA isolated from the oil gland secretory cells of peppermint (
Mentha x piperita
) (McCaskill, D. & Croteau, R.,
Planta,
197:49-56 [1995]), afforded, after extensive database comparisons, two clones having homologues of unknown function in plants and eubacteria, the sequences of which contained a motif with homology to the putative ATP-binding domain of the GHMP (galactokinase, homoserine kinase, mevalonate kinase, and phosphomevalonate kinase) family of metabolite kinases. This putative kinase gene from peppermint and its
E. coli
orthologue, when overexpressed in
E. coli,
yielded a recombinant enzyme that catalyzes the ATP-dependent phosphorylation of isopentenol monophosphate (IP) to IPP. Feeding experiments with IP and several other isoprenoid precursors, using isolated peppermint secretory cells, confirmed the phosphorylation of IP to IPP to be the last step in the DXP pathway.
SUMMARY OF THE INVENTION
In accordance with the foregoing, a cDNA encoding isopentenyl monophosphate kinase (IPK) from peppermint (
Mentha x piperita
) has been isolated and sequenced, and the corresponding amino acid sequence has been deduced. Accordingly, the present invention relates to isolated DNA sequences which code for the expression of isopentenyl monophosphate kinase, such as the sequence designated SEQ ID NO:1 which encodes an isopentenyl monophosphate kinase protein (SEQ ID NO:2) from peppermint (
Mentha x piperita
). Additionally, the present invention relates to isolated, recombinant isopentenyl monophosphate kinase proteins, such as the isolated, recombinant isopentenyl monophosphate kinase protein from peppermint (
Mentha x piperita
) (SEQ ID NO:2). In other aspects, the present invention is directed to replicable recombinant cloning vehicles comprising a nucleic acid sequence, e.g., a DNA sequence which codes for an isopentenyl monophosphate kinase, or for a base sequence sufficiently complementary to at least a portion of DNA or RNA encoding isopentenyl monophosphate kinase to enable hybridization therewith (e.g., antisense RNA or fragments of DNA complementary to a portion of DNA or RNA molecules encoding isopentenyl monophosphate kinase which are useful as polymerase chain reaction primers or as probes for isopentenyl monophosphate kinase or related genes). In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of isopentenyl monophosphate kinase, and the inventive concepts may be used to facilitate the production, isolation and purification of significant quantities of recombinant isopentenyl monophosphate kinase (or of its primary enzyme products) for subsequent use, to obtain expression or enhanced expression of isopentenyl monophosphate kinase in plants, microorganisms or animals, or may be otherwise employed in an environment where the regulation or expression of isopentenyl monophosphate kinase is desired for the production of this kinase, or its enzyme product, or derivatives thereof.


REFERENCES:
Sprenger, G.A. et al., “Identification of a thiamin-dependent synthase inEscherichia colirequired for the formation of the 1-deoxy-D-xylulose 5-phosphate precursor to isoprenoids, thiamin, and pyridoxol,”Proc. Natl. Acad. Sci USA, 94:12857-12862 (1997).
Lois, L.M. et al., “Cloning and characterization of a gene fromEscherichia coliencoding a transketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5-phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis,”Proc. Natl. Acad. Sci. USA, 95:2105-2110 (199

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