JP2014003938A - New enzyme and method for producing bacteriochlorophyll using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a bacteriochlorin compound having an ethylidene group at the C8 position, and an enzyme therefor.SOLUTION: In the method for producing a bacteriochlorin compound, BchX, BchY and BchZ derived from photosynthetic bacterium producing bacteriochlorophyll b or bacteriochlorophyll g are contacted with a chlorine compound having a vinyl group at the C8 position,thus a conjugated diene part of a B ring is 1,4-added and reduced to produce a bacteriochlorin compound having an ethylidene group at the C8 position.

Description

  The present invention relates to a method for producing a compound having an ethylidene group at the C8 position of a bacteriochlorin ring, using a photosynthetic bacterium producing bacteriochlorophyll b or g or proteins BchX, BchY and BchZ derived from the bacterium, and bacteriochlorophyll b. The present invention relates to novel proteins BchX, BchY and BchZ derived from photosynthetic bacteria.

In order to expand the use of renewable energy, it is necessary to proceed with the introduction of solar power generation. To that end, it is important to reduce the cost and mass production of solar cells. However, high-purity silicon, which is the current raw material for solar cells, is expensive and has a problem in securing its stability in Japan.
As a next-generation solar cell that replaces a silicon solar cell, a dye-sensitized solar cell that uses photo-induced electron transfer of a dye similar to the initial process of photosynthesis has attracted attention. So far, in addition to those using artificial porphyrins as sensitizing dyes, dye-sensitized solar cells using natural chlorophyll a derivatives have been reported. However, in order to increase the light energy conversion efficiency, it is desired to extend the absorption wavelength to a longer wavelength near infrared region.

Bacteriochlorophyll (BChl) is the name given to tetrapyrroles found in non-oxygen-producing photosynthetic bacteria. There are 7 types from a to g, and 6 types except BChl f so far. Has been found in nature. Among them, BChl a, BChl b and BChl g have a bacteriochlorin skeleton in which the CC double bond at the β-position of both the B and D rings of the tetrapyrrole ring is reduced to a single bond, while BChl c to f Has a chlorin skeleton in which only the D ring is reduced. BChl c / d / e is present in the light collecting part of chlorosome, which is an extramembranous antenna system of green sulfur bacteria, and forms a self-aggregate containing only a dye not involving protein. In contrast, BChla and BChlb are present in red photosynthetic bacteria, and BChlg is present in heliobacteria, forming non-covalent bonds with oligopeptides to form dye-protein complexes.
The absorption spectrum of (bacterio) chlorophyll has four absorption bands from the long wavelength side: Qy, Qx, By, and Bx (By and Bx bands are collectively referred to as the Soray band), but BChl a / has a bacteriochlorin skeleton. In b / g, the Qy band is greatly shifted in the near infrared region by a longer wavelength than BChl c / d / e and chlorophyll having a chlorin skeleton. In particular, BChl b has a longer wavelength shift in the Qy band than BChl a due to the presence of the ethylidene side chain at the C8 position. Therefore, BChl b, bacteriochlorophyllide b (BChlide b) from which the long-chain hydrocarbon group in the propionate residue on the 17th position is removed, bacteriopheophytin b (BPhe b) and bacteriopheophore from which the central metal is eliminated. If bide b (BPheoide b) can be supplied at low cost and in large quantities, it is useful for the production of optical devices with improved light energy conversion efficiency.

  However, in photosynthetic bacteria that produce BChl b or BChl g, the biosynthetic pathway for introducing an ethylidene group at the C8 position of the bacteriochlorin skeleton remains unclear. According to the conventional hypothesis, as in the case of BChla-producing bacteria, the chlorophyllide a reductase (COR) consisting of the three proteins chlorophyllide a (Chlide a) to BchX, BchY and BchZ is The double bond becomes a single bond (Non-Patent Document 1), and further, the C3 position is acetylated by the action of BchF and BchC to produce BChlide a, and then the ethyl group at the C8 position becomes an ethylidene group by the action of an unknown enzyme. Although predicted to be converted, a protein with enzymatic activity that catalyzes such a reaction has not been isolated from BChl b producing bacteria.

  On the other hand, it is possible to artificially introduce an ethylidene group onto the A ring of the tetrapyrrole ring by photoreduction, but there is no report that an ethylidene group was chemically introduced onto the B ring.

Nomata et al., J. Biol. Chem., 281 (21): 15021-15028 (2006)

  Therefore, an object of the present invention is to elucidate a biosynthetic pathway in which an ethylidene group is introduced into the C8 position of a bacteriochlorin ring in BChl b-producing bacteria and BChl g-producing bacteria, and to identify enzyme genes involved in the biosynthetic pathway The present invention provides an efficient method for producing BChl b or BChl g or a derivative thereof using the enzyme.

The present inventors include BChl b-producing bacteria and BChl g-producing bacteria that have an enzyme that reduces the 8-vinyl group of 8-vinylchlorophyllide a (8V-Chlide a) to an ethyl group. Noting the absence of 3,8-) divinylprotochlorophyllide a 8-vinyl reductase (DVR), BChl b producing bacteria and BChl g producing bacteria produce BChl g directly from 8V-Chlide a (ie chlorin). It was predicted that it would have an enzyme that catalyzes the reaction of adding 1,4-addition reduction of the conjugated diene of the ring B ring to give an ethylidene group at the C8 position of the bacteriochlorin ring.
Therefore, the present inventors isolated BchX, BchY and BchZ gene orthologs of BChla-producing bacteria from the genomic DNA of BChlb-producing Blastochloris viridis, subcloned into Escherichia coli, and BchX / Y / Z was expressed, 8V-Chlide a was reacted with this, and the reaction product was analyzed by HPLC. Surprisingly, BChlide g (long chain hydrocarbon group was bonded to position 17 to form BChl g It was found that a precursor was generated. Surprisingly, even when Chlide a was allowed to act on BchX / Y / Z derived from B. viridis, 3-vinyl bacteriochlorophyllide a (3V-BChlide a) was not produced. As a result of sequencing, BchX / Y / Z derived from B. viridis showed high sequence similarity to BchX / Y / Z derived from Rhodobacter capsulatus, a BChla-producing bacterium. It was totally unexpected to catalyze different reactions.
Furthermore, the present inventors searched for BchX, BchY and BchZ genes from known genomic sequences of Heliobacterium modesticaldum, a BChl g-producing bacterium, and identified the proteins encoded by these B. viridis. Similar to the derived BchX / Y / Z, the conjugated diene on the B ring of 8V-Chlide a was identified as having an enzyme activity that results in 1,4 addition reduction to produce BChlide g.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention provides the following.
[1] BchX, BchY and BchZ derived from photosynthetic bacteria producing bacteriochlorophyll b or bacteriochlorophyll g are represented by the following formula (1):

(Where
R ₁ represents a vinyl group, a 1-hydroxyethyl group or an acetyl group,
R ₂ represents a hydrogen atom, a C _1-18 alkyl group, a farnesyl group, a phytyl group, a geranylgeranyl group, a dihydrogeranylgeranyl group or a tetrahydrogeranylgeranyl group,
M does not exist or represents a divalent metal atom. )
A compound represented by the following formula (2):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
The manufacturing method of the compound represented by these.
[2] The method according to [1] above, wherein BchX, BchY and BchZ are obtained from a host cell transformed with an expression vector containing a nucleic acid encoding them.
[3] The above [1] or [2], wherein BchX, BchY and BchZ are derived from Blastochloris viridis, Thioflavicoccus mobilis or Heliobacterium modesticaldum ] The method of description.
[4] The method according to any one of [1] to [3], wherein M is a magnesium atom or a zinc atom.
[5] The protein according to any one of (a) to (c) below.
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: Along with the protein consisting of the amino acid sequence shown in 6, the following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and comprising the amino acid sequence shown in SEQ ID NO: 4 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
[6] A protein of any one of (a) to (c) below that catalyzes the reaction represented by:
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence represented by SEQ ID NO: 2; Along with the protein consisting of the amino acid sequence shown in 6, the following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and comprising the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
A protein that catalyzes the reaction represented by [7] Any one of the following proteins (a) to (c):
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and the amino acid sequence represented by SEQ ID NO: 2; Along with the protein consisting of the amino acid sequence shown in 4, the following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and consisting of the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 together with the following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
A protein that catalyzes the reaction represented by [8] a DNA encoding the protein of [5] above.
[9] DNA of the following (a) or (b).
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that comprises the amino acid sequence represented by SEQ ID NO: 4 and the protein that comprises the amino acid sequence represented by SEQ ID NO: 6 The following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by
[10] DNA encoding the protein according to [6] above.
[11] DNA of the following (a) or (b).
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 3
(b) together with a DNA complementary to the DNA of (a) above under stringent conditions and a protein comprising the amino acid sequence represented by SEQ ID NO: 2 and a protein comprising the amino acid sequence represented by SEQ ID NO: 6 The following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by
[12] DNA encoding the protein according to [7] above.
[13] DNA of the following (a) or (b).
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that has the amino acid sequence represented by SEQ ID NO: 2 and the protein that has the amino acid sequence represented by SEQ ID NO: 4 The following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by
[14] An expression vector containing one or more DNAs selected from the following (a) to (c).
(a) DNA according to [8] or [9] above
(b) DNA according to [10] or [11] above
(c) DNA according to [12] or [13] above
[15] A host cell transformed with the expression vector of [14] above.
[16] The host cell described in [15] above is cultured in a medium, and the resulting culture is selected from the protein described in [5] above, the protein described in [6] above and the protein described in [7] above A method for producing the protein, comprising recovering the above protein.
[17] The cell according to [15] above, wherein the host cell is a photosynthetic bacterium that produces bacteriochlorophyll a.

  According to the present invention, it is possible to stably provide BChl b or g or a derivative thereof in which the Qy band is in the near-infrared region of a longer wavelength, and therefore, functional dyes having a changed light absorption band are used industrially. It becomes possible to do.

It is a figure which shows alignment of the amino acid sequence of BchX derived from B. viridis and known BchX derived from other photosynthetic bacteria. R. caps: Rhodobacter capsulatus; R. spha: Rhodobacter sphaeroides; B. viridis: Blastochloris viridis; THIO. Mobilis: flaviocus mobilic ; H. modesti: Heliobacterium modesticaldum It is a figure which shows alignment of the amino acid sequence of BchY derived from B. viridis and known BchY derived from other photosynthetic bacteria. Abbreviations of bacterial names in the figure are the same as above. It is a figure which shows alignment of the amino acid sequence of BchZ derived from B. viridis and known BchZ derived from other photosynthetic bacteria. Abbreviations of bacterial names in the figure are the same as above. It is a figure which shows the absorption spectrum in 80-% acetone of the reaction product at the time of using 8-vinyl-Chlide a (A) or Chlide a (B) as a substrate (a residual substrate is included). The broken line in the figure is the control experiment result when BchX / Y / Z is not added, and the solid line is the absorption spectrum when reacted for 90 minutes according to the example. The horizontal axis of the graph indicates the wavelength (nm).

  The present invention relates to BchX, BchY and BchZ derived from photosynthetic bacteria that produce BChl b or BChl g, the following formula (1):

(Where
R ₁ represents a vinyl group, a 1-hydroxyethyl group or an acetyl group,
R ₂ represents a hydrogen atom, a C _1-18 alkyl group, a farnesyl group, a phytyl group, a geranylgeranyl group, a dihydrogeranylgeranyl group or a tetrahydrogeranylgeranyl group,
M does not exist or represents a divalent metal atom. )
By contacting a compound represented by the following formula (2):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
The method of manufacturing the compound represented by this is provided.
The enzyme composed of proteins BchX, BchY and BChZ derived from BChl b or BChl g producing bacteria used in the present invention (may be abbreviated as BchX / Y / Z) is C8 position represented by the formula (1) In addition, a 1,4-addition reduction of the conjugated diene part of the B ring of a chlorin compound having a vinyl group to the catalyst is carried out to convert it into a bacteriochlorin compound having an ethylidene group at the C8 position. The enzyme is a chlorophyllide a reductase (COR) activity possessed by BchX / Y / Z derived from a photosynthetic bacterium producing BChl a (eg, Rhodobacter capsulatus etc.), that is, the following formula (4):

It does not have an activity to catalyze the reaction represented by

  The photosynthetic bacteria derived from BchX, BchY and BchZ used in the present invention are not particularly limited as long as they produce BChl b or BChl g. Examples of BChl b producing bacteria include Blast chloris viridis, thio Flavivococcus mobilis, Thiococcus pfennigii, Thioalkalicoccus limnaeus, Blastochloris sulfoviridis, Halorhospira halochloris and HalorhodoBC halochloris Examples include Heliobacterium chlorum, Heliobacterium chlorum, Heliobacterium gestii, Heliophilum fasciatum, Heliobacillus mobilis ) Etc. It is below. Preferably, BchX, BchY and BchZ derived from B. viridis, T. mobilis and H. modern dam can be used.

  BchX, BchY and BchZ are (1) a method of extracting and purifying bacteria producing BChl b or BChl g as a raw material, (2) a method of chemically synthesizing, (3) BchX, BchY or BchZ by genetic recombination technology. By appropriately purifying from cells that have been manipulated to express, or (4) biochemically synthesizing from nucleic acids encoding BchX, BchY, or BchZ using a cell-free transcription / translation system, etc. Can be acquired.

  Isolation and purification of BchX, BchY, and BchZ from bacteria that naturally produce BChl b or BChl g (BChl b / g producing bacteria) can be performed, for example, as follows. BChl b / g-producing bacteria are homogenized in an appropriate buffer solution (Since photosynthetic bacteria are anaerobic bacteria, the operation is preferably performed using a solution degassed in an anaerobic chamber). A cell extract can be obtained by treatment with an active agent and the like, and can be purified by appropriately combining separation techniques conventionally used for protein separation and purification. Such separation techniques include, for example, methods utilizing differences in solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, non-denaturing polyacrylamide electrophoresis (PAGE), sodium dodecyl sulfate-poly A method using molecular weight difference such as acrylamide electrophoresis (SDS-PAGE), a method using charge such as ion exchange chromatography and hydroxyapatite chromatography, a method using specific affinity such as affinity chromatography, and the like Examples include, but are not limited to, a method utilizing a difference in hydrophobicity such as phase high performance liquid chromatography and a method utilizing a difference in isoelectric point such as isoelectric focusing. BchX, BchY and BchZ may be either purified or roughly purified. Alternatively, a crude extract of BChl b / g producing bacteria may be used.

Production of BchX, BchY, and BchZ by chemical synthesis is performed, for example, by synthesizing all or part of the sequence using a peptide synthesizer based on the amino acid sequences shown in SEQ ID NOs: 2, 4, and 6. be able to. The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. Producing a target protein by condensing a partial peptide or amino acid capable of constituting BchX, BchY and BchZ of the present invention with the remaining part and removing the protecting group when the product contains a protecting group; Can do. Here, the condensation and the removal of the protecting group are carried out according to a method known per se, for example, the method described in the following (1) and (2).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, NewYork (1965)

The BchX, BchY and BchZ of the present invention thus obtained can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
In the case where BchX, BchY and BchZ obtained by the above method are free forms, the free forms can be converted into an appropriate salt by a known method or a method analogous thereto, and conversely, a protein is obtained as a salt. In this case, the salt can be converted into a free form or other salt by a known method or a method analogous thereto.

  BchX, BchY and BchZ of the present invention are preferably isolated from a culture of a transformant containing an expression vector carrying the nucleic acid by cloning (or chemically synthesizing) the nucleic acid encoding the protein. Can be manufactured.

Cloning of enzyme genes is usually performed by the following method. First, the enzyme is completely or partially purified from a cell or tissue that produces the desired enzyme, and its N-terminal amino acid sequence is determined using the Edman method or mass spectrometry. In addition, the amino acid sequence of an oligopeptide obtained by partially decomposing the enzyme with a protease or chemical substance that cleaves the peptide in a sequence-specific manner is similarly determined by Edman method or mass spectrometry. An oligonucleotide having a nucleotide sequence corresponding to the determined partial amino acid sequence is synthesized, and this is used as a probe from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme. ) The DNA encoding the enzyme is cloned by a hybridization method.
Alternatively, an antibody against the enzyme is prepared according to a conventional method using all or part of the completely or partially purified enzyme as an antigen, and the antibody is prepared from a cDNA or genomic DNA library prepared from a cell or tissue producing the enzyme. DNA encoding the enzyme can also be cloned by a screening method.
When the gene of an enzyme similar in enzymatic properties to the target enzyme is known, for example, the NCBI BLAST website (http://www.ncbi.nlm.nih.gov/BLAST/) is accessed and the known gene Search for a sequence homologous to the nucleotide sequence of the gene, create a probe as described above based on the hit nucleotide sequence, and clone the DNA encoding the enzyme by colony (or plaque) hybridization be able to. For example, in the case of B. viridis-derived BchX, BchY and BchZ consisting of the respective amino acid sequences shown in SEQ ID NOs: 2, 4 and 6, B.chch, BchY and BchZ derived from H. At the amino acid level, with 38%, 33% and 37% identity (homology of 56%, 53% and 54%, respectively), the nucleotide sequences of B. viridis-derived BchX, BchY and BchZ genes ( By homology search with SEQ ID NOs: 1, 3 and 5), ORFs (SEQ ID NOs: 7, 9 and 11) encoding BchX, BchY and BchZ on the genome of H.
In addition, based on the hit nucleotide sequence, an appropriate oligonucleotide was synthesized as a primer, and a genomic DNA fraction prepared from BChl b / g-producing bacteria or a total RNA or mRNA fraction was used as a template for Polymerase Chain Reaction ( Hereinafter, it can also be directly amplified by “PCR method” or Reverse Transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”).
The nucleotide sequence of the DNA obtained as described above can be determined using a known sequence technique such as the Maxam-Gilbert method or the dideoxy termination method.

  The cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

The protein can be produced by transforming a host with an expression vector containing DNA encoding BchX, BchY or BchZ and culturing the resulting transformant.
An expression vector containing DNA encoding BchX, BchY or BchZ is, for example, excising the target DNA fragment from DNA encoding BchX, BchY or BchZ and ligating the DNA fragment downstream of the promoter in an appropriate expression vector. Can be manufactured. DNAs encoding BchX, BchY, and BchZ may be inserted into separate vectors, or two or more may be inserted into a single vector. For example, from the analogy with BchlX / Y / Z derived from BChla-producing bacteria with COR activity, BchX / Y / Z of the present invention is also a catalytic component that BchY / Z transfers electrons to the substrate and completes the reduction reaction. Since BchX can be predicted to function as a reducing component that transfers electrons to the catalytic component, DNA that encodes BchX alone is mounted on an expression vector, and DNA that encodes BchY and DNA that encodes BchZ Can be mounted on a separate expression vector. Since BchX / Y / Z is present in one operon on the genome of the BChl b / g-producing bacterium from which it is derived, a genomic sequence containing both BchY-encoding ORF and BchZ-encoding ORF is subcloned into an expression vector. By doing so, it is possible to design BchY / Z to be expressed polycistronically from a single promoter.
As expression vectors, plasmids derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13); plasmids derived from Bacillus subtilis (eg, pUB110, pTP5, pC194); yeast-derived plasmids (eg, pSH19, pSH15) are used. .
The promoter may be any promoter as long as it is appropriate for the host used for gene expression. For example, when the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.

  When a bacterium is used as a host cell, the expression vector generally needs to contain a replicable unit capable of autonomous replication in the host cell in addition to the promoter region and terminator region described above. The promoter region includes an operator and a Shine-Dalgarno (SD) sequence in the vicinity of the promoter. When yeast is used as the host, the expression vector preferably further contains an enhancer sequence, mRNA 5 'and 3' untranslated regions, a polyadenylation site, and the like.

As the host, for example, Escherichia, Bacillus, yeast or the like is used.
Examples of the genus Escherichia include, for example, Escherichia coli K12 / DH1 [Proc. Natl. Acad. Sci. USA, 60, 160 (1968)], JM103 [Nucleic Acids Research, 9, 309 (1981). ], JA221 [Journal of Molecular Biology, 120, 517 (1978)], HB101 [J. Mol. Biol., 41, 459 (1969)], C600 [Genetics, 39, 440 (1954)], etc. Is used. Examples of the Bacillus bacterium include Bacillus subtilis MI114 [Gene, 24, 255 (1983)], 207-21 [J. Biochem., 95, 87 (1984)]. Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris (Pichia pastoris) KM71 etc. are used.

  Transformation can be performed according to a known method depending on the type of host. Escherichia can be transformed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982). Bacillus can be transformed according to the method described in, for example, Molecular & General Genetics, 168, 111 (1979). Yeast can be transformed, for example, according to the method described in Methods in Enzymology, 194, 182-287 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).

The culture of the transformant can be performed according to a known method depending on the type of the host. For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for the growth of a transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose; examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5-8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, M9 medium containing glucose and casamino acid [Journal of Experiments in Molecular Genetics, 431-433, Cold Spring Harbor Laboratory, New York 1972] Is preferred. If necessary, an agent such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently.
Culturing of a transformant whose host is an Escherichia bacterium is usually performed at about 15 to 43 ° C. for about 3 to 24 hours. If necessary, aeration or agitation may be performed. The transformant whose host is Bacillus is usually cultured at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, aeration or agitation may be performed. As a medium for culturing a transformant whose host is yeast, for example, Burkholder minimum medium [Proc. Natl. Acad. Sci. USA, vol. 77, 4505 (1980)] or 0.5% casamino acid SD medium containing [Proc. Natl. Acad. Sci. USA, 81, 5330 (1984)]. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.
As described above, BchX, BchY and BchZ can be generated in the cells of the transformant.

BchX, BchY and BchZ can be separated and purified from the culture obtained by culturing the transformant according to a method known per se. For example, microbial cells collected from a culture by a known method are suspended in an appropriate buffer solution, and the microbial cells are disrupted by ultrasonic waves, lysozyme and / or freeze-thawing, and then a crude extract is obtained by centrifugation or filtration. A method or the like is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 ^™ .
BchX, BchY and BchZ contained in the crude extract thus obtained can be further purified according to a method known per se. Such methods include the use of solubilities such as salting out and solvent precipitation; mainly using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

  When BchX, BchY or BchZ thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and when the protein is obtained as a salt, The salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.

As a means for obtaining recombinant BchX / Y / Z quickly and conveniently, an amino acid sequence (for example, histidine, arginine) that can be adsorbed to a metal ion chelate at a certain portion (preferably N or C terminal) of the coding sequence of each protein. A DNA sequence encoding a basic amino acid sequence such as lysine, preferably a sequence consisting of histidine, is added by gene manipulation and expressed in a host cell, and the BchX / Y / Z active fraction of the culture of the cell From the above, a method of separating and recovering BchX / Y / Z by affinity with a carrier on which the metal ion chelate is immobilized is preferably exemplified. The metal ion chelate adsorbent used for purification is a transition metal, for example, a divalent ion of cobalt, copper, nickel, iron, or a trivalent ion of iron, aluminum, etc., preferably a solution containing a divalent ion of cobalt or nickel. A ligand, for example, iminodiacetic acid (IDA) group, nitrilotriacetic acid (NTA) group, tris (carboxymethyl) ethylenediamine (TED) group, etc. is contacted with the attached matrix to bind to the ligand. The matrix part of the chelate adsorbent is not particularly limited as long as it is a normal insoluble carrier.
Alternatively, affinity purification can be performed using glutathione-S-transferase (GST), maltose binding protein (MBP), HA, FLAG peptide, Strep tag peptide, or the like as a tag.

In another preferred embodiment of the present invention, a transformant that recombinantly produces BchX, BchY, and BchZ derived from BChl b / g-producing bacteria can be produced using BChla-producing bacteria as host cells. In general, there are many BChla-producing bacteria that are easier to culture than BChl b / g-producing bacteria, have relatively short generation times, and can be cultured at high density. Useful. For example, pJN3 and pJNY constructed from the broad host range vector pBBR1-MCS2 can be mentioned as large-scale expression vectors in R. capsulatus. Both vectors control the expression of pucAB encoding a light-harvesting bacteriochlorophyll-binding protein, and have a cloning site downstream of the puc promoter that exhibits high transcriptional activity under anaerobic conditions, allowing insertion of DNA encoding the target protein. . Between the puc promoter and the cloning site, a 6xHN tag is inserted in pJN3 and a Strep tag is inserted in pJNY, facilitating purification of the expressed protein. As a drug resistance marker, a spectinomycin resistance gene has been newly introduced in addition to the kanamycin resistance gene derived from pBBR1-MCS2.
Transformation of R. capsulatus (introduction of an expression vector) utilizes conjugation with E. coli (a triple parental conjugation of donor E. coli containing the expression vector, helper E. coli that induces conjugation, and recipient R. capsulatus). Can be done. The donor E. coli is not particularly limited, and a normal strain such as JM105 may be used. As the helper Escherichia coli, XL1-Blue, DH5, C600 etc. having the helper plasmid pDPT51 can be used. As R. capsulatus, DB176 or the like in which the puc promoter is more strongly induced than the wild strain may be used.

When DNA encoding BchX, BchY and BchZ derived from BChl b / g is introduced into BChl a-producing bacteria such as R. capsulatus, the resulting transformant is capable of producing BChl b / g in addition to BChl a To have. Here, homologous recombination can be used to inactivate the DVR and transformants can be converted to BChl b / g bacteria. In particular, such a transformant can improve the yield due to microbial production of BChl b / g because the product can be converted to BChl b / g while retaining the high BChl producing ability of BChla-producing bacteria. It is feasible.
In particular, R. Capsulatus makes a phage-like particle called GTA, but when GTA overproduction mutant strains (eg, R121, Y262, CB1127 strain) are used, the targeting vector is introduced into the mutant strain by conjugation with E. coli and cultured. A large amount of GTA accumulates in the medium, and some of these have cut out the targeting vector and incorporated it into the particles. Therefore, when this culture filtrate is brought into contact with the recipient's R. capsulatus, the targeting vector is introduced from the GTA into the recipient cell and homologous recombination is induced, so that the target gene is efficiently targeted to the target locus. be able to.

Using BchX / Y / Z derived from BChl b / g producing bacteria produced by any of the above methods, a reaction to introduce an ethylidene group at the C8 position of the bacteriochlorin compound (1,4 of the conjugated diene part of the B ring) (Addition reduction reaction) is performed as follows.
The compound serving as a substrate for the reaction is represented by the following formula (1):

(Where
R ₁ represents a vinyl group, a 1-hydroxyethyl group or an acetyl group,
R ₂ represents a hydrogen atom, a C _1-18 alkyl group, a farnesyl group, a phytyl group, a geranylgeranyl group, a dihydrogeranylgeranyl group or a tetrahydrogeranylgeranyl group,
M does not exist or represents a divalent metal atom. )
If it is a compound represented by this, it will not specifically limit.
Here, examples of the “C _1-18 alkyl group” include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, hexyl, octyl, caprylyl, lauryl, palmityl, stearyl and the like.
The central metal “M” includes, for example, a divalent metal atom such as a magnesium atom, a zinc atom or a copper atom, preferably a magnesium atom or a zinc atom. No natural BChl b / g contains a zinc atom as a central metal, but it is more stable than magnesium and is suitable for industrial use of the dye.
The compound represented by the formula (1) can be produced chemically by a method known per se, or by culturing mutant photosynthetic bacteria lacking some BChl biosynthetic enzymes. Some compounds are commercially available.

The 1,4-addition reduction reaction of conjugated dienes is carried out in the appropriate buffer solution (for example, phosphate buffer, Tris buffer, borate buffer, HEPES buffer, etc.), the above substrate, BchX / Y / Z, ATP It can be carried out by mixing divalent metal ions or the like at an appropriate concentration and incubating in the dark under anaerobic conditions. The substrate concentration is, for example, 0.1 to 100 μM, preferably 1 to 50 μM, more preferably 5 to 10 μM. The amount of BchX / Y / Z added is, for example, 0.001 to 20 mg / ml, preferably 0.01 to 2 mg / ml, more preferably 0.02 to 0.5 mg / ml. The concentration of ATP added is, for example, 0.1 to 10 mM, preferably 1 to 5 mM, more preferably 1 to 2 mM. Examples of the divalent metal ions, a MgCl _2, ZnCl _2, CuCl _2, etc., for example 0.1 to 20 mM, preferably can be added 2 to 10 mM, more preferably 5 to 6 mM.

It is desirable to further add dithiothreitol, phosphocreatine, creatine phosphokinase, dithionite, Triton X-100 ^™, etc. to the reaction solution.

  The reaction is desirably performed within a range of a suitable temperature and a suitable pH of BchX / Y / Z. For example, the reaction temperature is 10 to 50 ° C, preferably 20 to 45 ° C. Moreover, as pH of a reaction liquid, it is pH 6-10, for example, Preferably it is 7-9. The reaction time is not particularly limited, and is, for example, 10 minutes to 24 hours, preferably 30 minutes to 6 hours.

  The following formula (2) generated by the reaction:

(In the formula, R ₁ , R ₂ , and M are as defined above.)
The compound represented by is obtained by extracting the reaction solution with an organic solvent such as diethyl ether or acetone, and using an analytical method such as reverse phase HPLC or LC / MS to obtain a fraction that gave a peak corresponding to the product. It can be separated and purified by recovery.

  The present invention also provides the following formula (3):

(In the formula, R ₁ , R ₂ , and M are as defined above.)
A novel protein BchX / Y / Z that catalyzes the reaction represented by:

BchX of the present invention is any one of the following proteins (a) to (c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: A protein that catalyzes the reaction represented by formula (3) together with a protein consisting of the amino acid sequence shown in FIG.
(c) A protein having the identity of 95% or more with the amino acid sequence represented by SEQ ID NO: 2 and comprising the amino acid sequence represented by SEQ ID NO: 4 and the protein comprising the amino acid sequence represented by SEQ ID NO: 6, Protein that catalyzes the reaction represented by 3) BchY of the present invention is one of the following proteins (a) to (c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence represented by SEQ ID NO: 2; A protein that catalyzes the reaction represented by formula (3) together with a protein consisting of the amino acid sequence shown in FIG.
(c) A protein having the identity of 95% or more with the amino acid sequence shown in SEQ ID NO: 4 and consisting of the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 together with the formula ( Protein that catalyzes the reaction represented by 3) BchZ of the present invention is one of the following proteins (a) to (c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and the amino acid sequence represented by SEQ ID NO: 2; A protein that catalyzes the reaction represented by formula (3) together with the protein consisting of the amino acid sequence shown in FIG.
(c) A protein having the identity of 95% or more with the amino acid sequence shown in SEQ ID NO: 6 and comprising the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 together with the formula ( Protein that catalyzes the reaction represented by 3)

  In the above (b), “several” means 2, 3, 4, 5, or 6.

  The identity / homology of the amino acid sequence in (c) above is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gaps; matrix) = BLOSUM62; Filtering = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms are NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program It includes built-in 'or the like, may they likewise preferably used.

The present invention also provides DNAs encoding the above novel BchX, BchY and BchZ proteins.
The DNA encoding the novel BchX of the present invention is preferably the following DNA (a) or (b).
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that comprises the amino acid sequence represented by SEQ ID NO: 4 and the protein that comprises the amino acid sequence represented by SEQ ID NO: 6 DNA encoding a protein that catalyzes the reaction represented by formula (3)
The DNA encoding the novel BchY of the present invention is preferably the following DNA (a) or (b).
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 3
(b) together with a DNA complementary to the DNA of (a) above under stringent conditions and a protein comprising the amino acid sequence represented by SEQ ID NO: 2 and a protein comprising the amino acid sequence represented by SEQ ID NO: 6 DNA encoding a protein that catalyzes the reaction represented by formula (3)
The DNA encoding the novel BchZ of the present invention is preferably the following DNA (a) or (b).
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that has the amino acid sequence represented by SEQ ID NO: 2 and the protein that has the amino acid sequence represented by SEQ ID NO: 4 DNA encoding a protein that catalyzes the reaction represented by formula (3)

  In the above (b), the “stringent condition” means that nucleotide sequences having high identity, for example, nucleotide sequences having 95%, 97%, or 98% or more identity are hybridized and more identical. Under conditions where nucleotide sequences with low specificity do not hybridize, specifically, washing conditions for normal Southern hybridization, 60 ° C., 1 × SSC, 0.1% SDS, preferably 0.1 × SSC, 0.1% SDS, more preferably 68 Examples include conditions of washing once, more preferably 2 to 3 times at a salt concentration and temperature corresponding to ° C., 0.1 × SSC, and 0.1% SDS.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these in any meaning.

Example 1 Isolation of B. viridis-derived BchX, BchY and BchZ genes DNA containing the nucleotide sequence of SEQ ID NO: 1 and the nucleotide sequences of SEQ ID NOs: 3 and 5 at the same time using the genomic DNA of B. viridis as a template Amplified.
As a primer corresponding to the 5 'end
5′-ATGGTAACCTGCATTAGCGCCATGACGTCCCCCGCCAGAGC-3 ′ (SEQ ID NO: 13)
As a primer corresponding to the 3 'end side,
Using 5′-ATGGTAACCTGCATTATATCACACCGTGTCGTAAATCACTTCGA-3 ′ (SEQ ID NO: 14), a DNA containing the nucleotide sequence shown in SEQ ID NO: 1 (hereinafter referred to as bchX) by PCR reaction, and a primer corresponding to the 5 ′ end side
5′-ATGGTAGGTCTCAGCGCCATGAGGGCTGCCAGTTACGTTC-3 ′ (SEQ ID NO: 15),
As a primer corresponding to the 3 'end side,
Using 5′-ATGGTAGGTCTCATATCACGCAGCCTGCCCCCCGACA-3 ′ (SEQ ID NO: 16), DNA containing the nucleotide sequences shown in SEQ ID NOS: 3 and 5 (hereinafter referred to as bchY and bchZ, respectively) was amplified by PCR reaction (PCR conditions: 94 ° C-2 minutes 30 seconds 1 cycle, 94 ° C-30 seconds, 60 ° C-30 seconds, 72 ° C-3 minutes 30 seconds 35 cycles, 72 ° C-8 minutes, 4 ° C-∞ 1 cycle.TOYOBO KOD -plus-). Primer removal and buffer replacement were performed on the reaction solution containing DNA containing bchX and bchY-bchZ amplified by PCR reaction using NucleoSpin Extract II kit (Macherey-Nagel).
The obtained DNA fragment containing bchX was treated with the restriction enzyme BspMI (New England BioLabs), the DNA fragment containing bchY-bchZ was treated with the restriction enzyme BsaI (New England BioLabs), and pASK- treated with BsaI in the same manner. It was inserted into IBA5plus (manufactured by IBA) to obtain pA5-BvX and pA5-BvYZ, respectively. The obtained plasmid was sequenced, and it was confirmed that bchX and bchY-bchZ amplified by PCR had no error. 1-1 to 1-3 show alignment of amino acid sequences of BchX, BchY and BchZ derived from B. viridis and known BchX, BchY and BchZ derived from other photosynthetic bacteria.
pA5-BvX and pA5-BvYZ were separately introduced into E. coli Rosetta2 (Novagen), which is an expression host, to obtain transformants.

Example 2 Enzymatic activities of B. viridis-derived BchX / Y / Z and R. capsulatus-derived BchX / Y / Z (1) Expression in E. coli
Transformants into which pA5-BvX and pA5-BvYZ were respectively introduced were pre-cultured in 100 mL of LB medium. The entire amount of the preculture solution cultured overnight at 37 ° C in the dark was added to 1.2 L of LB medium, and cultured at 37 ° C in the dark for 2 hours. Thereafter, anhydrotetracycline was added to a final concentration of 200 μg / L, and cultured in the dark at 20 ° C. for 6 hours to express a large amount of the target protein.
(2) Activity measurement
In 250 mL of 100 mM HEPES-KOH buffer (pH 8.0), 5 mM MgCl ₂ , 5 mM dithiothreitol, 2 mM ATP, 20 mM phosphocreatine, 21 units / ul creatine phosphokinase, 0.7 mM dithionite, 0.05% Triton X-100 ^TM , 5 mM Chlide a or 5 mM 8-vinyl-Chlide a as substrate, purified BchX 0.1 mg / mL, co-purified BchY and BchZ added to 0.02 mg / ml, respectively The reaction was performed in an anaerobic chamber for 90 minutes at 34 ° C in the dark. After the reaction time has elapsed, the reaction is stopped by adding 1 mL of acetone to the reaction solution to denature the protein, and centrifugation is performed using a tabletop centrifuge to precipitate the denatured protein. It was collected. An equivalent amount of hexane was added to the supernatant fraction, and the mixture was stirred well, centrifuged with a table centrifuge, the lower acetone / water layer was collected, and the absorption spectrum was measured using a spectrophotometer. FIG. 2 shows the absorption spectrum of the reaction product (including residual substrate) in 80% acetone when each substrate is used. The broken line in the figure is the control experiment result when BchX / Y / Z is not added, and the solid line is the absorption spectrum when reacted for 90 minutes according to the example. When 8-vinyl-Chlide a was used as a substrate, the absorption peak derived from the substrate near 666 nm decreased, and BChlide g having a new peak at 756 nm was generated (FIG. 2A). On the other hand, when Chlide a is used as a substrate, even if BchX / Y / Z derived from B. viridis is mixed with Chlide a, only the absorption peak (666 nm) derived from the substrate decreases, and there is no reaction product. Did not occur.

By using BchX / Y / Z derived from the BChl b / g producing bacterium of the present invention, it is possible to introduce an ethylidene group on the C8 position of the bacteriochlorin skeleton. The dyes obtained in this way have the longest absorption wavelength maximum shifted to the near infrared region compared to other chlorins or bacteriochlorin compounds. By using this as a dye sensitizer, the light energy conversion efficiency is higher than before. There is a possibility that a dye-sensitized solar cell excellent in the above can be provided.
In addition, cancer cells actively divide and proliferate and easily take up fat-soluble porphyrin compounds having a large π plane, so porphyrin compounds can be used to distinguish cancer cells from normal cells. Attempts have been made to use. However, there is a problem that visible light cannot reach the cancerous part because the living body pigment such as heme absorbs the part deep from the cell surface. A bacteriochlorin compound having an ethylidene group at the C8 position has an absorption band in the near-infrared region of a longer wavelength, and is therefore useful for deep cancer diagnosis.
Furthermore, it has been clarified that chlorophyll molecules also have an anticancer effect, and research is being conducted as photodynamic therapy. Therefore, a C8 ethylidene bacteriochlorin compound having a strong absorption band in the near infrared region that is selectively adsorbed to cancer cells can be a useful tool for cancer treatment.

Claims (17)

BchX, BchY and BchZ derived from photosynthetic bacteria producing bacteriochlorophyll b or bacteriochlorophyll g are represented by the following formula (1):
(Where
R ₁ represents a vinyl group, a 1-hydroxyethyl group or an acetyl group,
R ₂ represents a hydrogen atom, a C _1-18 alkyl group, a farnesyl group, a phytyl group, a geranylgeranyl group, a dihydrogeranylgeranyl group or a tetrahydrogeranylgeranyl group,
M does not exist or represents a divalent metal atom. )
A compound represented by the following formula (2):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
The manufacturing method of the compound represented by these.   The method according to claim 1, wherein BchX, BchY and BchZ are obtained from a host cell transformed with an expression vector containing a nucleic acid encoding them.   The method according to claim 1 or 2, wherein BchX, BchY and BchZ are derived from Blastochloris viridis, Thioflavicoccus mobilis or Heliobacterium modesticaldum.   The method according to any one of claims 1 to 3, wherein M is a magnesium atom or a zinc atom. Protein of any of the following (a)-(c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2
(b) a protein comprising the amino acid sequence shown in SEQ ID NO: 2 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence shown in SEQ ID NO: 4 and SEQ ID NO: Along with the protein consisting of the amino acid sequence shown in 6, the following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and comprising the amino acid sequence shown in SEQ ID NO: 4 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by Protein of any of the following (a)-(c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 4
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 4 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and consisting of the amino acid sequence represented by SEQ ID NO: 2; Along with the protein consisting of the amino acid sequence shown in 6, the following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 4 and comprising the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 6 (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by Protein of any of the following (a)-(c).
(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 6
(b) a protein comprising the amino acid sequence represented by SEQ ID NO: 6 consisting of an amino acid sequence in which 1 to several amino acids are substituted, deleted, inserted or added, and the amino acid sequence represented by SEQ ID NO: 2; Along with the protein consisting of the amino acid sequence shown in 4, the following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by
(c) A protein having 95% or more identity with the amino acid sequence shown in SEQ ID NO: 6 and consisting of the amino acid sequence shown in SEQ ID NO: 2 and the protein consisting of the amino acid sequence shown in SEQ ID NO: 4 together with the following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
Protein that catalyzes the reaction represented by   DNA encoding the protein according to claim 5. The following DNA (a) or (b):
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 1
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that comprises the amino acid sequence represented by SEQ ID NO: 4 and the protein that comprises the amino acid sequence represented by SEQ ID NO: 6 The following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by   DNA encoding the protein according to claim 6. The following DNA (a) or (b):
(a) DNA comprising the nucleotide sequence represented by SEQ ID NO: 3
(b) together with a DNA complementary to the DNA of (a) above under stringent conditions and a protein comprising the amino acid sequence represented by SEQ ID NO: 2 and a protein comprising the amino acid sequence represented by SEQ ID NO: 6 The following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by   DNA encoding the protein according to claim 7. The following DNA (a) or (b):
(a) DNA consisting of the nucleotide sequence represented by SEQ ID NO: 5
(b) A protein that hybridizes under stringent conditions with a DNA complementary to the DNA of (a) above, and that has the amino acid sequence represented by SEQ ID NO: 2 and the protein that has the amino acid sequence represented by SEQ ID NO: 4 The following formula (3):
(In the formula, R ₁ , R ₂ , and M are as defined above.)
DNA encoding a protein that catalyzes the reaction represented by An expression vector containing one or more DNAs selected from the following (a) to (c).
(a) DNA according to claim 8 or 9
(b) DNA according to claim 10 or 11
(c) DNA according to claim 12 or 13   A host cell transformed with the expression vector according to claim 14.   Culturing the host cell according to claim 15 in a medium, and recovering one or more proteins selected from the protein according to claim 5, the protein according to claim 6, and the protein according to claim 7 from the obtained culture. A method for producing the protein, comprising:   The cell according to claim 15, wherein the host cell is a photosynthetic bacterium that produces bacteriochlorophyll a.