Study on the photochemical reaction of actinorhodopsin and its mediated phototrophy in the natural host

• Material Type: 博士論文
• Title: Study on the photochemical reaction of actinorhodopsin and its mediated phototrophy in the natural host
• Author/Editor: 中村, 槙太郎
• Author Heading: 中村, 槙太郎
• Publication, Distribution, etc.: Hokkaido University
• Publication Date: 2017-03-23
• Publication Date (W3CDTF): 2017-03-23
• Alternative Title: アクチノロドプシンの光化学反応と宿主細胞における機能的発現に関する研究
• Contributor: 出村, 誠; 姚, 閔; 菊川, 峰志
• Degree grantor/type: 北海道大学
• Date Granted: 2017-03-23
• Date Granted (W3CDTF): 2017-03-23
• Dissertation Number: 甲第12724号
• Degree Type: 博士(生命科学)
• Conferring No. (Dissertation): 甲第12724号
• Text Language Code: eng
• NDC: 400
• Target Audience: 一般
• Note (General): Microbial rhodopsins are transmembrane proteinsconsisted of seven α-helixes. They are found in various microorganisms and bind to an all-transretinal chloromophore. Bacteriorhodopsin (BR) is the first microbial rhodopsin which was discovered from thecytoplasmic membrane of the haloarchaeon Halobacterium salinarum. Since then, several microbial rhodopsins were found but they were mainly restricted to the Haloarchaea before 2000. However, metagenomic analysis revealed a new species of microbialrhodopsin from the gene of γ-Proteobacteria from Monterey Bay. This new rhodopsin was named Proteorhodopsin(PR). PR functions as a light-driven outward H+pump like BR and PR-like genes have been identified from numerous oceans microorganisms to date. H+-pumping rhodopsins such as BR or PR use light energy to generate a concentration gradient of proton across the membrane that is used for ATP synthesis. This fact indicates that most of organisms in marine environments use a rhodopsin to take in sunlight energy.On the other hand, for non-marine aqueous environments, PR-like rhodopsin genes were also revealed.This was named actinorhodopsin (ActR) because thesegenes areexclusively associated with actinobacteria. ActR was predicted H+-pump rhodopsin from amino acid sequence and its genes were indeed found in cultivated freshwater actinobacteriaand further identified in other freshwater bacterioplankton. It is established that ActR genes are widespread in freshwater environments, so most of organisms in all aquatic environmentstake in energy from sunlight using rhodopsins.In part I of this thesis, Idemonstrate ActR phototrophy in the native actinobacterium.Genome analysis showed that CandidatusRhodoluna planktonicastrain MWH-Dar1, a freshwater actinobacterium, encodes one microbial rhodopsin (RpActR) belonging to the ActR family. Reflecting the functional expression of RpActR, illumination induced the acidification of the actinobacterial cell suspension and then elevated the ATP content inside the cells.I indicated that MWH-Dar1is the first actinobacterium confirmed to performActR phototrophy.In part II of this thesis, I demonstrate photochemical charactazation of RpActR.The photochemistry of RpActR was also examined using heterologously expressed RpActR in Escherichia colimembrane. The purified RpActR showed λmaxat 534 nmand the pKa of the proton acceptor(Asp92) was determined to be 5.8.This value is ratherdifferent from BR but is similar to the members of the xanthorhodopsin(XR)-like family.However, unlike XR-like family, the donor mutantof RpActR showed almost the same pKa value for wild-type RpActR. This indicates that RpActR showsonly weak coupling between the donor and the acceptorunlike XR. I also investigated a photocycle characterization.The photocycle of RpActR underwent by the very fast formation of M intermediate. This means that the fast H+transfer between proton Schiffbase and proton acceptor residueoccurs and might be caused by their specific coordination in the photolyzed state.The subsequent intermediate, named P620, could be assigned to the O intermediate in other H+pumps. In contrast to conventional O, the accumulation of P620remains prominent, even at high pH. I considered that acertain residue might deprotonate at alkaline pH, which in turnelevates the pKa of the acceptor residue. Flash-induced absorbance changes suggested that there exist only one kind of photocycle at any pH. However, above pH 7, RpActR shows heterogeneity in the H+transfer sequences: one first captures H+and then releases it during the formation and decay of P620, while the other first releases H+prior to H+uptake during P620formation.This heterogeneity might confer a survival advantage to the cells in the freshwater environment. In this thesis, Ipresent the first observation of ActR phototrophy innativeactinobacterial cellsand photochemical characterization. The meaning of the unique photocycle reactions (such as heterogeneityor the decay of P620, the fast formation of M) should be clarified in future investigation.; (主査) 教授 出村 誠, 教授 姚 閔, 講師 菊川 峰志; 生命科学院（生命科学専攻）
• DOI: 10.14943/doctoral.k12724; https://doi.org/10.14943/doctoral.k12724
• Persistent ID (NDL): info:ndljp/pid/11039724; https://dl.ndl.go.jp/pid/11039724
• Collection: 障害者向け資料
• Collection (Materials For Handicapped People:1): テキストデータ
• Collection (particular): 国立国会図書館デジタルコレクション > デジタル化資料 > 博士論文; https://dl.ndl.go.jp/collections/A00014
• Acquisition Basis: 博士論文（自動収集）
• Date Accepted (W3CDTF): 2018-02-03T13:25:44+09:00
• Date Created (W3CDTF): 2017-03
• Format (IMT): PDF
• Access Restrictions: 国立国会図書館内限定公開
• Service for the Digitized Contents Transmission Service: 図書館・個人送信対象外
• Availability of remote photoduplication service: 可
• Periodical Title (URI): http://dx.doi.org/10.14943/doctoral.k12724; http://hdl.handle.net/2115/68026
• Is Referenced By (URI): http://hdl.handle.net/2115/65550
• Data Provider (Database): 国立国会図書館 : 国立国会図書館デジタルコレクション; https://dl.ndl.go.jp

• Summary, etc.: Microbial rhodopsins are transmembrane proteinsconsisted of seven α-helixes. They are found in various microorganisms and bind to an all-trans...