FORMATION OF THE ISOCYCLIC RING OF CHLOROPHYLL BY ISOLATED CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS
FORMATION OF THE ISOCYCLIC RING OF CHLOROPHYLL BY ISOLATED CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS
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DOI：
10.1007/bf00042968
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发表时间：
1995-02-01
影响因子：
3.7
通讯作者：
BEALE, SI
中科院分区：
文献类型：
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作者：
BOLLIVAR, DW;BEALE, SI
莱茵衣藻叶绿体催化了氯合成s -腺苷- l-蛋氨酸的两个连续步骤：mg -原卟啉IX甲基转移酶和mg -原卟啉IX单甲基酯氧化环化酶。构建了一株具有细胞壁缺陷且在黑暗中不能形成叶绿素的双突变株。用BioNeb喷雾器在裂解质膜但不裂解叶绿体包膜的条件下破坏暗生长细胞。用Percoll密度梯度离心纯化叶绿体。纯化后的叶绿体用于确定从mg -原卟啉TX生物合成mg -2,4-二乙烯基卟啉a(5)（二乙烯基原叶绿内酯）所需的组分。产物的形成需要添加mg -原卟啉IX，这是s -腺苷- l-蛋氨酸的底物：mg -原卟啉IX甲基转移酶，该酶产生mg -原卟啉IX单甲基酯。原位生成的mg -原卟啉IX单甲基酯是mg -原卟啉IX单甲基酯氧化环化酶的底物。通过离子对反相柱和聚乙烯柱的激发发射荧光光谱法和高效液相色谱法鉴定了反应产物为mg -2,4-二乙烯基卟啉a(5)（二乙烯基原叶绿素内酯）。偶联酶系统生成mg -2,4-二乙烯基卟啉a(5)需要O-2，并通过NADP（+）、NADPH再生系统和s -腺苷- l-蛋氨酸的加入来刺激。产品以相对稳定的速度形成至少60分钟。
Chlamydomonas reinhardtii chloroplasts catalyzed two sequential steps of Chi biosynthesis, S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase and Mg-protoporphyrin IX monomethyl ester oxidative cyclase. A double mutant strain of C. reinhardtii was constructed which has a cell wall deficiency and is unable to form chlorophyll in the dark. Dark-grown cells were disrupted with a BioNeb nebulizer under conditions which lysed the plasma membrane but not the chloroplast envelope. Chloroplasts were purified by Percoll density gradient centrifugation. The purified chloroplasts were used to define components required for the biosynthesis of Mg-2,4-divinylpheoporphyrin a(5) (divinyl protochlorophyllide) from Mg-protoporphyrin TX. Product formation requires the addition of Mg-protoporphyrin IX, the substrate for S-adenosyl-L-methionine:Mg-protoporphyrin IX methyltransferase which produces Mg-protoporphyrin IX monomethyl ester. The Mg-protoporphyrin IX monomethyl ester that is generated in situ is the substrate for Mg-protoporphyrin IX monomethyl ester oxidative cyclase. The reaction product was identified as Mg-2,4-divinylpheoporphyrin a(5) (divinyl protochlorophyllide) by excitation and emission spectrofluorometry and HPLC on ion-paired reverse-phase and polyethylene columns. Mg-2,4-divinylpheoporphyrin a(5) formation by the coupled enzyme system required O-2 and was stimulated by the addition of NADP(+), an NADPH regenerating system, and S-adenosyl-L-methionine. Product was formed at a relatively steady rate for at least 60 min.