Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fmicb.2019.00495

Web of Science

Scopus

PubMed

Language：Japanese   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：Japanese   Publishing type：Research paper (scientific journal)  

DOI： 10.7554/eLife.39028

Web of Science

Scopus

PubMed

Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Nature Publishing Group  

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a key enzyme to produce chlorophyll in the dark. Among photosynthetic eukaryotes, all three subunits chlL, chlN, and chlB are encoded by plastid genomes. In some gymnosperms, two codons of chlB mRNA are changed by RNA editing to codons encoding evolutionarily conserved amino acid residues. However, the effect of these substitutions on DPOR activity remains unknown. We first prepared cyanobacterial ChlB variants with amino acid substitution(s) to mimic ChlB translated from pre-edited mRNA. Their activities were evaluated by measuring chlorophyll content of dark-grown transformants of a chlB-lacking mutant of the cyanobacterium Leptolyngbya boryana that was complemented with pre-edited mimic chlB variants. The chlorophyll content of the transformant cells expressing the ChlB variant from the fully pre-edited mRNA was only one-fourth of the control cells. Co-purification experiments of ChlB with Strep-ChlN suggested that a stable complex with ChlN is greatly impaired in the substituted ChlB variant. We then confirmed that RNA editing efficiency was markedly greater in the dark than in the light in cotyledons of the black pine Pinus thunbergii. These results indicate that RNA editing on chlB mRNA is important to maintain appropriate DPOR activity in black pine chloroplasts.

DOI： 10.1038/s41598-017-02630-2

Web of Science

Scopus

PubMed

DOI： 10.1016/j.bbabio.2013.06.007

PubMed

DOI： 10.1038/srep01217

PubMed

DOI： 10.1093/pcp/pcr132

PubMed

There are two structurally unrelated protochlorophyllide (Pchlide) reductases in photosynthetic organisms; one is dark-operative reductase (DPOR) and the other is light-dependent reductase (LPOR). Since angiosperms employ LPOR as the sole Pchlide reductase, seedlings in the dark accumulate Pchlide that binds to LPOR to form prolamellar body (PLB) characterized by a unique paracrystalline structure in etiolplasts. However, it is unclear if LPOR has an intrinsic property to form PLB and how LPOR have evolved to form PLB during evolution. Here we show aberrant structure formation by overexpression of a cyanobacterial LPOR in a mutant lacking DPOR of the cyanobacterium <i>Leptolyngbya boryana</i>. We isolated a transformant overexpressing the <i>L. boryana</i> LPOR. Aberrant structures apparently different from thylakoid were observed in the dark grown cells by electron microscopy. LPOR localization in the aberrant structures is supported by immunocytochemical microscopy with anti-LPOR antiserum. Further cellular and biochemical analysis suggested that the cyanobacterial LPOR forms the aberrant structures but fails to form a photoactive complex.

DOI： 10.14841/jspp.2011.0.0685.0

Dark-operative protochlorophyllide reductase (DPOR) is a nitrogenase-like enzyme consisting of two components L-protein (ChlL) and NB-protein (ChlN-ChlB) and plays a critical role in chlorophyll (Chl) biosynthesis in the dark. All subunits of DPOR are encoded in plastid genome in photosynthetic eukaryotes. RNA editing in <I>chlN</I> and <I>chlB</I>, which is required for the restoration of codons for evolutionarily conserved amino acid residues, was reported in some gymnosperms. Here we report that RNA editing regulates the DPOR activity in black pine <I>Pinus thunbergii</I>. We constructed a series of shuttle vectors to express <I>P. thunbergii chlN-chlB</I> with and without mutations responsible for the RNA editing, and introduced them into the mutants lacking <I>chlB</I> of the cyanobacterium <I>Leptolyngbya boryana</I>. Only the transformants carrying the edited <I>chlN-chlB</I> genes restored the ability of Chl biosynthesis in the dark. Biochemical analysis of the cyanobacterial NB-protein with site-directed mutations responsible for the RNA editing supported the <I>in-vivo</I> complementation result. These results suggested that RNA editing regulates the greening in the dark through the NB-protein activity in <I>P. thunbergii</I>.

DOI： 10.14841/jspp.2011.0.0173.0

Dark-operative protochrolophyllide reductase (DPOR) is a nitrogenase-like enzyme consisting of three subunits, ChlL, ChlN and ChlB, and plays a critical role in chlorophyll (Chl) biosynthesis in the dark. While nitrogenase is distributed only among some prokaryotes, there has been no direct evidence for functional operation of nitrogenase-like enzyme in any eukaryotes. Here we report functional expression of probable DPOR components encoded by the chloroplast genome of <I>Physcomitrella patens</I> in the cyanobacterium <I>Leptolyngbya boryana</I>. We constructed two shuttle vectors for overexpression of two DPOR components, ChlL and ChlN-ChlB from <I>P. patens</I>, and introduced them into the cyanobacterial mutants lacking <I>chlL</I> and <I>chlB</I>. Both transformants restored the ability of Chl biosynthesis in the dark, indicating that these chloroplast genes encode functional DPOR subunits and that each DPOR component is compatible between <I>P. patens</I> and <I>L. boryana</I>. This compatibility was also confirmed by <I>in vitro</I> reconstitution with purified ChlL and ChlN-ChlB.

DOI： 10.14841/jspp.2010.0.0107.0

DOI： 10.1093/pcp/pcp111

PubMed

Dark-operative protochrolophyllide reductase (DPOR) is a nitrogenase-like enzyme consisting of three subunits, ChlL, ChlN and ChlB, and plays a critical role in chlorophyll biosynthesis in the dark. DPOR is distributed widely not only in anoxygenic photosynthetic bacteria but also in oxygenic phototrophs, which raises a question how the nitrogenase-like DPOR operates in oxygenic photosynthetic cells. Here we report functional expression of cyanobacterial DPOR compornents in the cyanobacterium <I>Leptolyngbya boryana</I> to examine the oxygen sensitivity. We constructed plasmids for overexpression of ChlL and ChlN-ChlB. We detected unambiguous DPOR activity by mixing two crude extracts of <I>L. boryana</I> overexpressing ChlL and ChlN-ChlB, allowing to evaluating the activities of L-protein and NB-protein individually. L-protein activity in the extract was almost lost upon the exposure to oxygen for 10 min, while NB-protein activity was maintained after 60 min exposure. These results suggest that L-protein is the target of oxygen inactivation in cyanobacterial cells.

DOI： 10.1039/b802427h

PubMed

Dark-operative protochlorophyllide (Pchlide) reductase (DPOR) is a nitrogenase-like enzyme catalyzing the D-ring reduction of Pchlide in the penultimate step of chlorophyll biosynthesis in most oxygenic photosynthetic organisms. DPOR is the determinant enzyme for greening in the dark of the photosynthetic organisms. How the oxygen-sensitive enzyme operates in oxygenic photosynthetic cells remains unsolved. Here we report the functional expression of the cyanobacterial DPOR catalytic component, NB-protein (ChlN-ChlB), in <I>E. coli</I>. The <I>chlN</I> and <I>chlB</I> genes from the cyanobacterium <I>Leptolyngbya boryana</I> (formerly <I>Plectonema boryanum</I>) were artificially connected to form a small operon, <I>chlN-chlB</I>, in the overexpression plasmid. ChlN protein with Sterp-tag was co-purified with ChlB by an affinity column from the soluble fraction of <I>E. coli</I>. The addition of thus purified NB-protein to a crude extract of the LPOR-lacking mutant of <I>L. boryana</I> obviously stimulated the DPOR activity, indicating that an active form of NB-protein was expressed in <I>E. coli</I>.

DOI： 10.14841/jspp.2008.0.0520.0

Dark-operative protochlorophyllide oxidoreductase (DPOR) is a nitrogenase-like enzyme catalyzing D-ring reduction of protochlorophyllide to form chlorophyllide. DPOR consists of two components, L-protein and NB-proteins, which are structurally related to nitrogenase Fe-protein and MoFe-protein, respectively. Because functional expression of nitrogenase components requires a set of proteins unique in nitrogen-fixing organisms for assemble of metallocenters and maturation of the components, nitrogenase components are not expressed as active forms in conventional expression system of <I>Escherichia coli</I>. There has been no report examining if active DPOR components are expressed in <I>E. coli</I> due to the absence of reliable assay system. Here we report the successful functional expression of DPOR components in <I>E. coli</I>, which was evaluated by the assay system of <I>Rhodobacter capsulatus</I>. The results suggested that a set of proteins in <I>E. coli</I> supports assemble of metallocenters and maturation of DPOR components, which is different from nitrogenase components.

DOI： 10.14841/jspp.2007.0.461.0

Event date： 2024.3

Language：English   Presentation type：Poster presentation  

Venue：神戸  

Event date： 2024.3

Language：English   Presentation type：Oral presentation (general)  

Event date： 2024.3

Language：English   Presentation type：Oral presentation (general)  

Venue：神戸  

Event date： 2023.9

Language：English   Presentation type：Poster presentation  

Event date： 2023.9

Language：English   Presentation type：Poster presentation  

Event date： 2023.9

Language：English   Presentation type：Poster presentation  

Event date： 2023.3

Language：Japanese   Presentation type：Oral presentation (general)  

Venue：東北大学   Country：Japan  

空気中の窒素分子をアンモニアへと還元する酵素ニトロゲナーゼは、一部の原核生物に
保存され、窒素固定により地球上の窒素循環に大きく寄与している。ニトロゲナーゼには、ニトロゲナーゼ類似酵素（NFL ; Nitrogen fixation like）と呼ばれるホモログが存在している。NFLは、クロロフィル生合成（光合成）、メタン生成（炭素代謝）、メチオニン代謝（硫黄代謝）等の窒素固定とは異なる代謝経路において報告されており、原核微生物における嫌気的環境での基盤的な代謝を支える酵素群として知られている。我々は、紅色非硫黄細菌 Rhodobacter capsultus において未だ機能が不明なNFL (NflH, D, K)について機能解析を進めている。R. capsulatusにおけるnflH, D, K遺伝子はスルホン酸の一種であるタウリン代謝に関わる遺伝子クラスター近傍に位置し、培地へのタウリン及びイセチオン酸等のスルホン酸の添加により、その発現量が100倍以上増加することが示された。またnfl破壊株は、イセチオン酸を唯一の硫黄源とする培地において、光合成従属栄養条件で生育できないことが確認された。この結果は、このNFLがイセチオン酸代謝経路において必須の因子であることを示唆している。

Event date： 2023.3

Language：English   Presentation type：Poster presentation  

Venue：東北大学   Country：Japan  

Event date： 2022.12

Language：Japanese   Presentation type：Poster presentation  

Country：Japan  

Event date： 2022.8

Language：English   Presentation type：Poster presentation  

Venue：Liverpool, UK   Country：United Kingdom  

Event date： 2022.8

Language：English   Presentation type：Poster presentation  

Venue：Liverpool, UK   Country：United Kingdom  

Event date： 2022.7

Language：Japanese   Presentation type：Oral presentation (invited, special)  

Venue：東京都立大学南大沢キャンパス   Country：Japan  

海洋性シアノバクテリアAcaryochlorisはクロロフィル dを主要光合成色素として生合成するシアノバクテリアで、1996年にパラオ海域において初めて単離された。その後、世界中の様々な場所からAcaryochlorisが単離報告され、その生育環境が海洋以外にも多岐に渡ることが報告されている。今日最も実験材料とされているタイプストレインA. marina MBIC11017株はこれまで発見されたAcaryochlorisの中で唯一集光アンテナタンパク質フィコビリソームを保持している。このことからAcaryochlorisのoriginでは失われたフィコビリソームをA. marina MBIC11017は再獲得したことが予想される。さらに別のAcaryochlorisでは窒素固定酵素ニトロゲナーゼを保持し、実際に窒素固定的な生育が確認された例も報告された。これら両者の形質獲得には多数の遺伝子を獲得する必要があり、これらの遺伝子の水平伝播がどのように起こったのか興味深い。本研究ではタイプストレインA. marina MBIC11017と同じ海域で単離されたA. marina MBIC10699株のゲノム解析をおこなった。MBIC10699株は6.4 Mbのクロモソームと4つの巨大なplasmid (pREC1-4; 393, 329, 303, 205 kbp) から構成され、MBIC10699株はフィコビリソームは保持しない株であるが、解読されたゲノム情報をMBIC11017株と比較するとANIが98%で統計的にも非常に近い近縁種であることが分かった。このことからこれら2株の比較解析によってフィコビリソームの再獲得がどのように起こり、またフィコビリソームの再獲得により光化学系の遺伝子にどのような影響を与えたのかを議論する。また2株が保持するplasmidの構成および保存遺伝子の多様性から、巨大なplasmidを遺伝子プールとした大規模な遺伝子クラスターの獲得についても考察する。

Event date： 2022.3

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2022.3

Language：Japanese   Presentation type：Oral presentation (general)  

Country：Japan  

Event date： 2019.3

Language：Japanese   Presentation type：Oral presentation (invited, special)