Biochemical Characterization of Chlorophyllide a Reductase and Bacteriochlorophyll Synthase of Rhodobacter sphaeroides
- Subject Rhodobacter sphaeroides , Synechocystis , chlorophyllide a reductase , bacteriochlorophyll synthase , superoxide , competitive inhibition
- Publisher 서강대학교 일반대학원
- Adviser 이정국
- Issued 2010
- Awarded 2010. 2
- Thesis degree 박사
- Major 일반대학원 생명과학과
- URI entity http://www.dcollection.net/handler/sogang/000000045648
- Language 영어
- Rights 서강대학교의 논문은 저작권에 의해 보호받습니다
Abstract
Chlorophyllide a reductase (COR) of Rhodobacter sphaeroides, which were reconstituted with the purified subunits of BchX, BchY, and BchZ, reduced ring B of chlorophyllide a using NADH under anaerobic conditions. Interestingly, suppressor mutations rescuing the inability of R. sphaeroides FeSOD mutant to grow in succinate-based minimal medium were predominantly mapped to BchZ subunit of COR. The enzyme is labile in the presence of O2. However, it generates superoxide at low O2. The enzymes reconstituted with BchX, BchY, and the mutein subunit of BchZ from suppressor mutants showed less activity not only for chlorophyllide a reduction but also for superoxide generation compared with the enzyme reconstituted with the wild-type subunits. BchX, which contains FMN, and BchY are iron-sulfur proteins, whereas BchZ is a hemoprotein containing b-type heme. Neither chlorophyllide a reduction nor superoxide generation was observed with the enzyme reconstituted with the wild-type subunits of BchX and BchY, and the apo-subunit of BchZ that had been refolded without heme, in which FMN of BchX was fully reduced. Thus, superoxide is generated not from FMN of BchX but from heme of BchZ. Consistently, the heme of BchZ muteins was half reduced in its redox state compared with that of wild-type BchZ. The photosynthetic growth of Synechocystis sp. PCC6803 was ceased by the expression of R. spaheroides COR. However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely rescued the growth cessation. Thus, COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight for the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.
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The photosynthetic growth of Synechocystis sp. PCC6803 is hampered by the exogenously added bacteriochlorophyllide (Bchlide) a in a dose dependent manner. The growth inhibition caused by Bchlide a, however, is relieved by an increased level of exogenously added chlorophyllide (Chlide) a. The results are explained by the competitive inhibition of chlorophyll synthase by Bchlide a with KI of 0.3 mM and 1.14 mM in the presence of sufficient geranylgeranyl pyrophosphate (GGPP) and phytyl pyrophosphate (PPP), respectively. Surprisingly, the bacteriochlorophyll synthase of Rhodobacter sphaeroides is inhibited competitively by Chlide a with KI of 0.54 mM and 0.77 mM in the presence of sufficient GGPP and PPP, respectively. Consistently, the exogenously added Chlide a inhibits the metabolic conversion of the exogenously added Bchlide a to bacteriochlorophyll a by an R. sphaeroides bchFNB-bchZ mutant that neither synthesizes nor metabolizes Chlide a. The metabolic inhibition by Chlide a, however, is relieved by the elevated level of Bchlide a. Thus, the chlorophyll synthase of Synechocystis sp. PCC6803 and the bacteriochlorophyll synthase of R. sphaeroides, both of which perform ping-pong type reactions, are inhibited by Bchlide a and Chlide a, respectively. Although neither of inhibitors is catalyzed by target enzyme, inhibitions in the competitive mode suggest a structural similarity between their active sites.
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BchX, BchY, 및 BchZ 단량체들로 구성되는 Rhodobacter sphaeroides 유래의 chlorophyllide a reductase (COR)는 혐기조건에서 NADH를 사용하여 chlorophyllide a 의 B ring을 환원시킨다. 놀랍게도, 최소 배지상에서 성장하지 못하는 R. sphaeroides FeSOD 결손 균주의 성질이 COR의 BchZ 단량체 상에 발생하는 suppressor mutation에 의해 회복되었다. 이 효소는 산소 존재 시 비가역적으로 파괴되는 성질을 가지나, 낮은 산소 분압에서는 superoxide를 생성하는 것으로 나타났다. 시험관 내에서 재결합된 BchX, BchY, 및 변이된 BchZ는 야생형 효소복합체에 비해 감소된 chlorophyllide a 환원 활성 및 superoxide 생성 활성을 보였다. FMN을 그 조효소로 갖는 BchX, 그리고 BchY 단량체는 iron-sulfur center를 함유하는 반면, BchZ 단량체는 b-type의 heme을 함유하는 것으로 분석되었다. Heme을 가해주지 않은 상태에서 refolding된 BchZ는 BchX 및 BchY 단량체와 재조합하여도 chlorophyllide a 환원 활성 및 superoxide 생성 활성을 나타내지 못했다. 이 반응 조건에서 BchX의 FMN은 완전히 환원되어 있는 것으로 미루어보어, superoxide는 BchZ의 heme에 3 의해 발생하는 것으로 추정할 수 있었다. 변이된 여러 BchZ 단량체들을 시험한 결과, 환원된 heme의 비율이 야생형 BchZ에 비해 절반 정도로 낮은 것을 확인할 수 있었고 이는 효소 활성의 감소 정도와 일치하였다. Synechocystis sp. PCC6803의 광합성 성장은 R. sphaeroides 유래 COR의 발현에 의해 억제되었으나, 이러한 성장 억제는 세포질 내 superoxide dismutase의 활성을 증가시킴으로서 완전하게 회복되었다. 따라서, COR은 Synechocystis sp. PCC6803 내에서 superoxide를 발생시키며, 산소발생 광합성 균주 내의 산소 분압은 COR이 작용할 수 있을 만큼 충분히 낮은 것으로 나타났다. 이러한 Synechocystis sp. PCC6803의 성장 억제 현상은 bacteriochlorophyll a 생합성 경로로부터 COR 반응을 거치지 않는 chlorophyll a 생합성 경로로의 진화 과정을 설명할 수 있는 착안점을 제시해준다.
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