Hexulose phosphate isomerase gene

Details Hexulose phosphate isomerase gene Hexulose phosphate isomerase gene

1998-11-04

2001-12-18

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

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

Isomerase

C435S320100, C435S325000, C435S252300, C536S023100, C536S023200

Reexamination Certificate

active

06331428

ABSTRACT:

TECHNICAL FIELD
The present invention relates to a DNA coding for hexulose phosphate isomerase, and a method for producing hexulose phosphate isomerase.
BACKGROUND ART
The ribulose monophosphate pathway is known for C1 metabolism of methylotroph bacteria. This pathway comprises three stages starting from fixation of formaldehyde with ribulose 5-phosphate, followed by cleavage of fructose 6-phosphate and regeneration of ribulose 5-phosphate. The ribulose monophosphate pathway is a pathway which is coupled to several metabolic systems. The gene structures of respective enzymes involved in this pathway have been of great interest. However, few reports have been submitted on this pathway, and little genetic analysis has been achieved.
3-Hexulose-6-phosphate synthase (hereinafter referred to as “HPS”, if necessary), which catalyzes the initial reaction of the ribulose monophosphate pathway, has been already purified from
Methylomonas aminofaciens
which is a Gram-negative obligate methanol-assimilating bacterium. A gene coding for this enzyme has been cloned, and its primary structure has been reported (Yanase, H. et al., FEMS
Microbiol. Lett
., 135, 201-205 (1996)).
By the way, the biochemical substance, in which a specific position on the objective compound molecule is labeled with a stable isotope element, i.e., carbon 13 (
13
C), is useful for the study of metabolic pathway of the organism. Recently, the investigation of the situation of metabolic substances in vivo based on the use of the carbon 13-NMR is an extremely important technique for diagnosis of various diseases and daily health examination. It is necessary, or it has been demanded, for such a new technique, to inexpensively supply a substance in which a certain objective position is labeled with carbon 13.
The present inventor has been established a method for preparing [1-
13
C]D-glucose 6-phosphate from carbon 13-labeled methanol by utilizing the formaldehyde-fixing pathway of a methanol-assimilating bacterium. However, the yield of synthesis of the objective compound has not been so high (
Biosci. Biotech. Biochem
., 5, 308-312 (1993)).
Therefore, it has been demanded to obtain a system which makes it possible to efficiently produce only the objective product. A method is conceived as one of such systems, in which a series of respective enzymes for synthesizing labeled D-fructose 6-phosphate by using labeled formaldehyde and ribulose 5-phosphate are prepared, and a reaction system is constructed based on the use of the enzymes to efficiently prepare the objective labeled compound. As for HPS which is the initial enzyme in the reaction system, its gene has been isolated from
Methylomonas aminofaciens
as described above, and its structure has been also clarified. However, hexulose-phosphate isomerase (hereinafter referred to as “HPI” or phospho-3-hexuloisomerase (PHI), if necessary), which is an enzyme to catalyze the next step reaction, has been purified only partially, and neither its amino acid sequence nor gene structure has been known. Further, neither amino acid sequence nor gene structure has been reported for both enzymes of HPS and HPI of Gram-positive bacteria including the genus
Mycobacterium
as facultative methanol-assimilating bacteria.
In the circumstance as described above, in order to establish a system for efficiently preparing an objective labeled compound, it has been demanded to isolate a gene coding for HPI and provide a method for efficiently producing HPI based on the use of the gene.
DISCLOSURE OF THE INVENTION
The present invention has been made taking the foregoing viewpoints into consideration, an object of which is to provide a DNA coding for HPI and a method for utilizing the DNA.
The present inventor has been investigated the ribulose monophosphate pathway, especially HPS and its gene in
Mycobacterium gastri
which is a Gram-positive facultative methanol-assimilating bacterium. During this investigation, the present inventor has unexpectedly found a gene coding for HPI of this strain. Thus, the present invention has been completed.
That is, the present invention lies in a DNA coding for a protein as defined in the following (A) or (B):
(A) a protein which has an amino acid sequence depicted in SEQ ID NO: 13 in Sequence Listing; or
(B) a protein which comprises an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acids in the amino acid sequence depicted in SEQ ID NO: 13 in Sequence Listing, and which has a hexulose phosphate isomerase activity.
Specifically, the DNA described above includes DNA as defined in the following (a) or (b):
(a) a DNA which contains a nucleotide sequence corresponding to at least nucleotide numbers of 608 to 1204 of a nucleotide sequence depicted in SEQ ID NO: 12 in Sequence Listing; or
(b) a DNA which is hybridizable with the nucleotide sequence corresponding to at least nucleotide numbers of 608 to 1204 of the nucleotide sequence depicted in SEQ ID NO: 12 in Sequence Listing under a stringent condition, and which codes for the protein having the hexulose phosphate isomerase activity.
In another aspect, the present invention provides a cell into which the DNA is introduced in a form capable of expressing hexulose phosphate isomerase encoded by the DNA.
In still another aspect, the present invention provides a method for producing hexulose phosphate isomerase, comprising the steps of cultivating the cell in a medium to produce and accumulate the hexulose phosphate isomerase in a culture, and collecting the hexulose phosphate isomerase from the culture.
In the present invention, the term “hexulose phosphate isomerase activity” refers to an activity to catalyze an isomerization reaction between 3-hexulose 6-phosphate and fructose 6-phosphate.
The present invention will be explained in detail below.
<1>DNA of the Present Invention
The DNA of the present invention has been obtained from chromosomal DNA of
Mycobacterium gastri
as follows, as explained in Examples described later on.
At first, HPS is purified from
Mycobacterium gastri
MB19 strain. HPS can be purified from a cell-free extract solution of MB19 strain up to give a single band on SDS-PAGE by means of DEAE-sepharose column chromatography, phenylsepharose column chromatography, and DEAE-sepharose column chromatography. In each of the purification steps, the HPS activity can be measured in accordance with the method described in
Methods in Enzymology
, Vol. 188, 397-401 (1990).
Next, partial amino acid sequences of purified HPS are determined. Oligonucleotide primers to be used for PCR (polymerase chain reaction) are synthesized on the basis of the obtained amino acid sequence information to perform PCR by using a template of genomic DNA prepared from
Mycobacterium gastri
MB19 strain. The genomic DNA can be obtained in accordance with the method of Saito et al. (see
Biochim. Biophys. Acta
, 72, 619-629 (1963)). The cultivation to obtain bacterial cells used for preparing the genome is performed by using a medium containing methanol as a carbon source added with 1% of glycine. By doing so, a large amount of bacterial cells can be obtained, making it easy to perform the bacteriolysis operation. As for the bacteriolytic enzyme, sufficient bacteriolysis is not achieved, if only lysozyme is used. However, the use of N-acethylmuramidase SG is effective.
When oligonucleotides having nucleotide sequences shown in SEQ ID NO: 8 and SEQ ID NO: 9 in Sequence Listing are used as the primers, a DNA fragment of about 400 bp is obtained by means of PCR as described above.
Next, colony hybridization is performed for a library comprising PstI-digested fragments of the genomic DNA of
Mycobacterium gastri
MB19 strain by using, as a DNA probe, the DNA fragment amplified by PCR as described above so that a positive clone is obtained.
A nucleotide sequence has been determined for about 3 kb of a clone fragment having a length of about 4.1 kb obtained as described above. An obtained result is shown in SEQ ID NO: 12 in Sequence Listing.

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