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WO1997037028A2 - Ribose-5-phosphate isomerase (d-ribose-5-phosphate cetol isomerase, ec 5.3.1.6) - Google Patents

Ribose-5-phosphate isomerase (d-ribose-5-phosphate cetol isomerase, ec 5.3.1.6) Download PDF

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Publication number
WO1997037028A2
WO1997037028A2 PCT/EP1997/001539 EP9701539W WO9737028A2 WO 1997037028 A2 WO1997037028 A2 WO 1997037028A2 EP 9701539 W EP9701539 W EP 9701539W WO 9737028 A2 WO9737028 A2 WO 9737028A2
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WO
WIPO (PCT)
Prior art keywords
phosphate
ribose
phosphate isomerase
sequence
protein
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Application number
PCT/EP1997/001539
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German (de)
English (en)
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WO1997037028A3 (fr
Inventor
Ralf-Michael Schmidt
Jens Lerchl
Martin William
Claus Schnarrenberger
Josef Kellermann
Original Assignee
Basf Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Basf Aktiengesellschaft filed Critical Basf Aktiengesellschaft
Priority to AU25055/97A priority Critical patent/AU2505597A/en
Publication of WO1997037028A2 publication Critical patent/WO1997037028A2/fr
Publication of WO1997037028A3 publication Critical patent/WO1997037028A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/533—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving isomerase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)

Definitions

  • Ribose 5-phosphate isomerase (D-ribose 5-phosphate ketol isomerase, EC 5.3.1.6)
  • the present invention relates to proteins with Ribcse-5-phosphate isomerase activity, their use in test systems, and to nucleic acids which code for these proteins.
  • Plants are able to form organic compounds with the formation of oxygen using light energy from atmospheric carbon dioxide. This process is called photosynthesis.
  • the complex reactions that lead to carbon dioxide fixation are divided into light and dark reactions.
  • the light reaction serves to provide energy in the form of ATP and reduction equivalents in the form of NADPH.
  • the dark reaction reductive pentose phosphate cycle or Calvin cycle
  • these compounds are used for the synthesis of organic carbon compounds.
  • Some of the known herbicides e.g. dichlorophenylmethyl urea or paraquat
  • the dark reaction is not used as a target for herbicides.
  • the plastidic ribose 5-phosphate isomerase occupies a special position, which catalyzes the following reaction:
  • the enzyme has an amphibolic function, i.e. it is part of both the reductive pentosephosphate cycle (Calvin cycle) and the oxidative pentosephosphate pathway and thus has an important function in photosynthetic and non-photosynthetic tissues.
  • Ribose 5-phosphate is converted via the phosphoribosyl pyrophosphate synthetase into phosphoribosyl pyrophosphate, which acts as a ribose 5-phosphate donor molecule in tryptophan and nucleotide biosynthesis.
  • the erythrose-4-phosphate, sedoheptulose-7-phosphate, glyceraldehyde-3-phosphate and fructose are derived from the ribose-5-phosphate and ribulose-5-phosphate via the plastid transketolase and the ribulose-5-phosphate-3-epimerase - 6-phosphate.
  • Erythrose-4-phosphate is an intermediary between primary and secondary metabolism.
  • erythrose-4-phosphate flows into the shikimate path, which leads to the synthesis of aromatic amino acids and phenolic substances.
  • Exported triose phosphates serve as substrates for glycolysis and gluconeogenesis in the cytoplasm.
  • Fructose-6-phosphate is used as a precursor for the production of starch in the plastids.
  • Ribulose-5-phosphate is converted, depending on ATP, by the phosphoribulokinase into ribulose-1, 5-bisphosphate, which functions as the primary carbon dioxide acceptor.
  • the plastid ribose 5-phosphate isomerase is present as a homodimer with a relative molecular mass of approx. 53 kDa ⁇ Rutner,
  • the object of the present invention was to provide a vegetable 35 ribose-5-phosphate isomerase in pure form by cloning the corresponding gene.
  • amino acid sequence shown in SEQ ID NO 2 is based on the translation of the cDNA sequence shown in SEQ ID NO 1.
  • the protein shown in SEQ ID NO 2 is a so-called precursor protein consisting of 289 amino acids.
  • the mature protein arises from the precursor form by splitting off the chloroplastic där transit peptide, which consists of 50 amino acids according to an N-terminal sequencing of the mature protein.
  • Both the precursor protein and proteins derived therefrom by substitution, deletion or insertion of amino acids and which still have ribose 5-phosphate isomerase activity belong to the proteins according to the invention.
  • Substitution means the exchange of one or more amino acids by one or more other amino acids. So-called conservative exchanges are preferably carried out, in which the new amino acid has a similar property to the original amino acid, for example replacement of Glu by Asp, Val by Ile, Ser by Thr.
  • Deletion is the replacement of an amino acid with a direct link; preferred positions for deletions are the termini of the polypeptide and the links between the individual protein domains.
  • Inserts are insertions of amino acids into the polypeptide chain, whereby a direct bond is formally replaced by one or more amino acids.
  • Proteins which are formed from N-terminal shortenings by 20 to 100 amino acids from SEQ ID NO 2 are particularly preferred.
  • the invention further relates to nucleic acids which code for the abovementioned proteins.
  • Suitable nucleic acid sequences can be obtained by back-translating the polypeptide sequence according to the genetic code. Those codons are preferably used for this which are frequently used according to the organism-specific "codon usage". The codon usage can easily be determined on the basis of computer evaluations of other known genes of the organism in question.
  • ribose 5-phosphate isomerase If the plant ribose 5-phosphate isomerase is to be expressed, for example, in a bacterium, it is often advantageous to use the "codon usage" of the bacterium for the back translation.
  • the invention furthermore relates to vectors for producing transgenic plants which contain the nucleic acids coding for the ribose 5-phosphate isomerase according to the invention together with functional regulation signals.
  • Regulatory signals include the constitutive gene expression-promoting promoters such as the 35S CaMV promoter ⁇ Franck et al. , 1980, Cell 21, 285-294 ⁇ as well as termination signals such as the polyadenylation signal of the Ti plasmid pT ⁇ ACH5 ⁇ Gielen et al., 1984. EMBO J. 2, 835-846 ⁇ and translation enhancers such as the 5 'guide sequence from tobacco mosaic virus ⁇ Gallie et al. , 1987, Nucl. Acids Res. 15, 5 8693-8711 ⁇ .
  • the proteins according to the invention are particularly suitable for the identification of herbicidal active substances, in particular for the detection of riosbose-5-phosphate isomerase-specific inhibitors.
  • the proteins can be used, for example, in an enzyme test in which the activity of the rbose-5-phosphate isomerase is determined in the presence and absence of the active substance to be tested.
  • a comparison of the two activity determinations 5 can be used to make a qualitative and quantitative statement about the inhibitory behavior of the active substance to be tested.
  • the invention further relates to herbicides which can be identified using a test system described above.
  • the invention also consists in a process for the preparation of herbicidal compositions which inhibit a vegetable r ⁇ bose-5-phosphate isomerase, which is characterized in that known chemical compounds are tested in a test described above and those with an inhibitory effect are customary with 0 Carrier and auxiliary substances formulated.
  • the method reproducibly allows a large number of substances to be selectively selected from those with a high potency, in order to subsequently carry out further in-depth tests known to the person skilled in the art.
  • the activity of the ribose 5-phosphate isomerase was determined in 40 mM potassium phosphate pH 7.4, 5 mM magnesium chloride, 0.5 U ribose 5-phosphate isomerase, 1 U transketolase, 1 U triose phosphate Isomerase and 1 U glycerol-3-phosphate dehydrogenase, 240 mM NADH, 10 mM R ⁇ bose-5-phosphate.
  • R ⁇ bose-5-phosphate isomerase was obtained as a commercially available preparation (Sigma). The purification was carried out by ion exchange chromatography with DEAE 1 '- Fractogel (Merck) equilibrated in 10 mM potassium phosphate, pH 7.5, 10 mM ethylene diammotetraacetate (EDTA) and 10 mM 2-mercaptoethanol. After dialysis against column buffer, the protein was added to the column and eluted through a gradient of 0-0.3 M potassium chloride in column buffer. Fractions with R ⁇ bose-5-phosphate isomerase activity were concentrated using polyethylene glycol 20000 and then dialyzed against column buffer.
  • the native molecular weight was determined via HPLC gel filtration at a flow rate of 1.0 ml / mm over Bio-Sil TSK 250 (Merck). The latter material was equilibrated in 50 mM sodium sulfate, 20 mM sodium dihydrogen phosphate pH 6.8. Beta-galactosidase (465 kD), immunoglobulin G (150 kD), antigen-binding antibody fragment (Fab, 50 kD) and myoglobm (17 kD) were used as protein standards. The native molecular weight was determined by interpolation of the graph from the retention time against the logarithm of the molecular weight.
  • Purified protein was cleaved using cyanogen bromide or with endoprotemase LysC as described ⁇ Eckerskorn and Lott arrived, 1989, Chromatographia 28,
  • the resulting peptides were separated using a 2 x 125 mm Supersher 60 RP select B column (Merck). A flow rate of 200 ⁇ l / mm m and a 1% / mm gradient of trifluoroacetic acid (0.1% (v / v)) in water and trifluoroacetic acid (0.1% (v / v)) in acetonitrile were used .
  • the resulting peptides were degraded in an automatic Porton 3600 sequencer (Beckman) via amino terminal degradation ⁇ Edman and Begg, 1967, Eur. J. Biochem., 80-91 ⁇ and the amino acids were identified using the Microbore HPLC System Gold (Beckman) .
  • Cloning processes such as restriction cleavage, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, ligation approaches, transformation of E. coli cells, cultivation of bacteria, multiplication of phages and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) ⁇ Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6 ⁇ .
  • the PCR amplification of the R ⁇ bose-5-phosphate isomerase was carried out in a DNA thermal cycler from Perkm Elmer.
  • the oligonucleotides used are shown in Figure 5.
  • the reaction mixtures contained 8 ng / 1 double-stranded leaf-specific spinach cDNA, 0.5 ⁇ M of the corresponding oligonucleotides, 50 ⁇ M
  • Nucleotides (Pharmacia), 50 mM potassium chloride, 10 mM Tris-HCl (pH 8.3 at 25 ° C), 1.5 mM magnesium chloride, and 0.02 U / 1 Taq polymerase (Perkin Elmer).
  • the amplification conditions were set as follows: Addition temperature ' : 45 ° C, 1 min denaturation temperature: 92 ° C, 1 min elongation temperature: 72 ° C, 1 min number of cycles: 35
  • the above sequence of 119 base pairs was used as hybridization probe (Clal / Xhol fragment of the plasmid pPCRrpil), which was carried out with the aid of a “Multiprime DNA labeling System” (Amersham Buchler) in the presence of ⁇ - 32 P-dCTP (Amersham; specific activity 3000 Ci / mmol) were radioactively labeled according to the manufacturer's instructions.
  • the membranes were hybridized after prehybridization at 60 ° C.
  • the sequencing of recombinant DNA molecules was carried out with an automatic laser fluorescence DNA sequencer (A.L.F.) from Pharmacia using fluorescent
  • the DNA sequence of the longest cDNA sequence of the clone pRI12 is shown in SEQ ID N01 (Genbank Accession number L43068).
  • the 1118 base pair long cDNA clone pRI12 contains an open reading frame of 870 bases and codes for a protein with 239 amino acids.
  • Analysis of the polypeptide using the peptide sequences of the microsequencing showed that a sequence with typical characteristics of a chloroplastic transit peptide of 50 amino acids is present at the N-termmus of the protein.

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  • Proteomics, Peptides & Aminoacids (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

L'invention concerne une protéine à activité de ribose-5-phosphate isomérase, renfermant une séquence aminoacide représentant une séquence partielle d'au moins 100 aminoacides de SEQ ID No 2. L'invention concerne également des acides nucléiques codant pour cette protéine, ainsi que son utilisation pour l'identification de substances actives herbicides.
PCT/EP1997/001539 1996-03-29 1997-03-26 Ribose-5-phosphate isomerase (d-ribose-5-phosphate cetol isomerase, ec 5.3.1.6) WO1997037028A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU25055/97A AU2505597A (en) 1996-03-29 1997-03-26 Ribose-5-phosphate isomerase (d-ribose-5-phosphate ketol isomerase, ec 5.3.1.6)

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DE19612772.6 1996-03-29
DE19612772 1996-03-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100896968B1 (ko) * 2007-03-21 2009-05-14 건국대학교 산학협력단 라이보스 5-인산 이성화효소를 이용한 알로스의 제조방법
US7655770B1 (en) * 1998-04-29 2010-02-02 Monsanto Technology Llc Nucleic acid molecules and other molecules associated with the phosphogluconate pathway

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU1165713A1 (ru) * 1983-02-25 1985-07-07 Институт физиологии и биофизики растений АН ТаджССР Способ получени рибозо-5-фосфат изомеразы из листьев шпината

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
APEL TW ET AL: "The ribose 5-phosphate isomerase-encoding gene is located immediately downstream form that encoding murine immunoglobulin kabba" GENE, Bd. 156, 1995, AMSTERDAM NL, Seiten 191-197, XP002036260 in der Anmeldung erwähnt *
CHEMICAL ABSTRACTS, vol. 103, no. 17, 28.Oktober 1985 Columbus, Ohio, US; abstract no. 137700, IVANISHCHEV, V. V. ET AL: "Isolation of ribose 5-phosphate isomerase from spinach leaves" XP002036262 & SU 1 165 713 A (USSR) *
MARTIN W ET AL: "Microsequencing and cDNA cloning of the Calvin cycle/OPPP enzyme ribose-5-phosphate isomerase (EC 5.3.1.6) from spinach chloroplasts." PLANT MOL BIOL, FEB 1996, 30 (4) P795-805, NETHERLANDS, XP002036257 *
RUTNER A C: "SPINACH-D 5 PHOSPHO RIBOSE ISOMERASE PURIFICATION AND PROPERTIES OF THE ENZYME" BIOCHEMISTRY, 9 (1). 1970 178-184., XP002036259 in der Anmeldung erwähnt *
SCHNARRENBERGER C ET AL: "Enzymatic evidence for a complete oxidative pentose phosphate pathway in chloroplasts and an incomplete pathway in the cytosol of spinach leaves" PLANT PHYSIOLOGY (ROCKVILLE), 108 (2). 1995. 609-614., XP002036258 in der Anmeldung erwähnt *
SORENSEN KI ET AL: "Ribose catabolism of Escherichia coli: characterization of the rpiB gene encoding ribose phosphate isomerase B and of the rpiR gene, which is involved in regulation of rpiB expression" JOURNAL OF BACTERIOLOGY, Bd. 178, Nr. 4, Februar 1996, US, Seiten 1003-1011, XP002036261 in der Anmeldung erwähnt *
WOODRUFF WW 3D ET AL: "Inhibition of ribose-5-phosphate isomerase by 4-phosphoerythronate." J BIOL CHEM, JUL 10 1979, 254 (13) P5866-7, UNITED STATES, XP002043068 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7655770B1 (en) * 1998-04-29 2010-02-02 Monsanto Technology Llc Nucleic acid molecules and other molecules associated with the phosphogluconate pathway
KR100896968B1 (ko) * 2007-03-21 2009-05-14 건국대학교 산학협력단 라이보스 5-인산 이성화효소를 이용한 알로스의 제조방법

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AU2505597A (en) 1997-10-22

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