Phosphohexuloisomerase and gene therefor

Details Phosphohexuloisomerase and gene therefor Phosphohexuloisomerase and gene therefor

2001-07-05

2003-10-07

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

Chemistry: molecular biology and microbiology

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

Isomerase

C435S252300, C536S023200

Reexamination Certificate

active

06630341

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to phosphohexuloisomerase and a DNA coding for it. More precisely, the present invention related to phosphohexuloisomerase derived from a thermotolerant bacterium,
Bacillus brevis
, and a DNA coding for it.
2. Description of the Related Art
Among organisms that can utilize single carbon (C1) compounds such as methane and methanol as a carbon source (methylotrophs), there are known those having the ribulose monophosphate (RuMP) pathway as a pathway for metabolizing such compounds. Important key enzymes of this pathway are hexulose phosphate synthase (HPS, 3-hexulose-6-phosphate synthase), which catalyzes the initial reaction of the ribulose monophosphate pathway, and phosphohexuloisomerase (PHI, phospho-3-hexuloisomerase), which catalyzes the subsequent reaction.
By the way, biochemical substances in which specific position of a target compound molecule is labeled with a stable isotope, carbon 13 (
13
C), are useful for study of biological metabolic pathway. Furthermore, it has recently become a very important technique to investigate behaviors of metabolic products in living bodies by using carbon 13-NMR techniques in diagnosis of various diseases and daily health examination. For such novel techniques, it is necessary and desired to provide compounds labeled at a certain target position with carbon 13 at a low cost.
As one of systems for producing such target compounds as mentioned above, a method can be conceived, in which a series of enzymes are prepared for synthesizing labeled D-fructose 6-phosphate using labeled formaldehyde and ribulose 5-phosphate, and a target labeled compound is efficiently prepared in a reaction system utilizing the enzymes. Hexulose phosphate synthase, which is an enzyme initially acts in the reaction system, has been isolated from several kinds of microorganisms, and some of its characteristics have been elucidated. Such microorganisms include, for example,
Methylomonas capsulatus
(J. R. Quayle,
Methods in Enzymology,
188, p.314, 1990), Methylomonas M15 strain (
Methods in Enzymology,
188, p.319, 1990),
Methylomonas aminofaciens
77a strain (
Biochim. Biophys. Acta.,
523, p.236, 1978),
Mycobacterium gastri
MB19 (
Methods in Enzymology,
188, p.393, 1990), and
Acetobacter methanolicus
MB58 (
Methods in Enzymology,
188, p.401, 1990).
Further, as for phosphohexuloisomerase, it has been partially purified from
Methylomonas aminofaciens
77a strain (Agric. Biol. Chem., 41 (7), p1133, 1977), and a purified enzyme and a gene coding for it were isolated from a gram-positive facultative methanol assimilating bacterium,
Mycobacterium gastri
(Japanese Patent Laid-open Publication (Kokai) No. 11-127869).
Enzymes and proteins produced by thermotolerant bacteria are generally stable at a high temperature, and most of them are also stable against pH variation and organic solvents. Therefore, applications thereof have been highly developed as diagnostic agents, industrial catalysts and so forth. As a C1 metabolic system enzyme of thermotolerant methanol assimilating bacteria, only hexulose phosphate synthase has been purified from
Bacillus methanolicus
C1 strain (
Methods in Enzymology,
188, p.393, 1990), and its detailed structure and gene therefor are unknown. On the other hand, as for phosphohexuloisomerase of thermotolerant bacteria, not only the structure of enzyme protein and gene therefor, but also purification of the enzyme have not been reported.
SUMMARY OF THE INVENTION
The inventors of the present invention found that, in the course of cloning of a gene coding for hexulose phosphate synthase (henceforth also referred to as “hps”) of
Bacillus brevis
S1 strain, a gene coding for PHI (henceforth also referred to as “phi”) existed in the DNA fragment containing hps. And they isolated the phi gene, introduced this gene into an
Escherichia coli
cell, and examined activity of the expression product to confirm that the gene coded for PHI. Thus, they accomplished the present invention.
That is, the present invention provides the followings.
(1) A DNA coding for a protein defined in the following (A) or (B):
(A) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing,
(B) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion or addition of one or several amino acid residues and having phosphohexulose isomerase activity.
(2) The DNA according to (1), which is a DNA defined in the following (a) or (b):
(a) a DNA containing a nucleotide sequence consisting of at least the residues of nucleotide numbers 1149-1700 of the nucleotide sequence of SEQ ID NO: 1 shown in Sequence Listing,
(b) a DNA which is hybridizable with a nucleotide sequence consisting of at least the residues of nucleotide numbers 1149-1700 of the nucleotide sequence of SEQ ID NO: 12 shown in Sequence Listing under a stringent condition, and codes for a protein having phosphohexulose isomerase activity.
(3) A cell into which a DNA according to (1) or (2) is introduced in such a manner that phosphohexulose isomerase encoded by the DNA can be expressed.
(4) A method for producing phosphohexulose isomerase, comprising culturing the cell according to (3) in a medium to produce and accumulate phosphohexulose isomerase in culture, and collecting the phosphohexulose isomerase from the culture.
(5) A protein defined in the following (A) or (B):
(A) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing,
(B) a protein having the amino acid sequence of SEQ ID NO: 3 shown in Sequence Listing including substitution, deletion, insertion or addition of one or several amino acid residues and having phosphohexulose isomerase activity.
According to the present invention, a DNA coding for phosphohexuloisomerase is obtained, and this enables efficient production of that enzyme. As a result, it becomes possible to provide labeled substances that are important and required for medicine or biochemical basic research in large quantities at low cost.
PREFERRED EMBODIMENTS OF THE INVENTION
Hereafter, the present invention will be explained in detail.
<1> DNA of the Present Invention
The DNA of the present invention was found in the DNA fragment containing hps gene of
Bacillus brevis
S1 strain adjacent to and downstream from the hps gene, and it can be isolated and obtained from chromosomal DNA of
Bacillus brevis
. Specifically, as shown in the examples described later, the DNA of the present invention was obtained from chromosomal DNA of
Bacillus brevis
as follows.
First, HPS is purified from
Bacillus brevis
. As the
Bacillus brevis
, the
Bacillus brevis
S1 strain can be mentioned. This strain is subcultured at NCIMB (The National Collections of Industrial and Marine Bacteria) with the accession number of NCIMB12524.
HPS can be purified from cell free extract of the S1 strain by Q-Sepharose column chromatography, Buthyl-Toyopearl column chromatography and Superdex 200 column chromatography to such a degree that it can be detected as a single band in SDS-PAGE. In each purification step, HPS activity can be measured by the method described in
Methods in Enzymology
, vol. 188, 397-401 (1990).
A partial amino acid sequence of the purified HPS is determined, and oligonucleotide primers for PCR (polymerase chain reaction) are synthesized based on the obtained amino acid sequence information. Then, PCR is performed by using genomic DNA prepared from the
Bacillus brevis
S1 strain as a template. The genomic DNA can be obtained by the method of Saito et al. (described in
Biochim. Biophys. Acta,
72, 619-629 (1963)). If the oligonucleotides having the nucleotide sequences of SEQ ID NOS: 7 and 8 shown in Sequence Listing are used as primers, a DNA fragment of about 400 bp will be obtained by the above PCR.
Then, based on the nucleotide sequence of the hps fragment obtained as described above, a DNA fragment containing the hps gene in its full length is obtained from
Bacillus brevis
S1

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