Analyses of structures and reaction mechanisms of biosynthetic enzymes of pyrroloquinoline quinone

吡咯喹啉醌生物合成酶的结构及反应机制分析

• 批准号: 15580064
• 负责人: TOYAMA Hirohide
• 金额: $ 2.05万
• 依托单位: Yamaguchi University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15580064/
• 关键词: pyrroloquinoline quinone; PQQ; biosynthesis

Gene cluster involved in biosynthesis of pyrroloquinoline quinone, PQQ, had been determined as pqqABCDEFG. The mutant defected in pqqC produced a biosynthetic intermediate, and it was converted to PQQ in vitro by PqqC protein, which was expressed in Escherichia coli transformant and purified. This intermediate was purified and its structure was confirmed as 3a-(2-amino-2-carboxy-ethyl)-4,5-dioxio-4,5,6,7,8,9-hexahydro-quinone-7,9-dicarboxylic acid. The PqqC enzyme reaction required a protein factor (ActF) plus NADPH to show full enzyme activity, however, dithiothreitol (DTT) itself could be replaced. When mixed with PQQ, PqqC/D, a fusion protein of PqqC and PqqD, was eluted as a complex in gel filtration column chromatography, but separated each other in the presence of DTT. In the case of PqqC, part of the complex remained even in the presence of DTT. DTT has an ability to reduce PQQ, thus PqqC and PqqC/D might bind the oxidized PQQ tightly but not the reduced form so much. Association constant of PqqC or PqqC/D with PQQ was determined by measuring fluorescent quenching of the protein with titration of PQQ. Partially purified ActF showed NADPH oxidase activity in the presence of PQQ. It is suggested that DTT or ActF plus NADPH keeps PQQ in the reduced form and consequently enhances PQQ production by PqqC or PqqC/D in vitro. PqqD might work to lower the affinity of PqqC to the reduced form of PQQ. PqqE was expressed in E.coli and purified in the presence of DTT. When DTT was absent, PqqE seemed easy to be degraded. It is suggested that PqqE contained iron-sulfur cluster according to its absorption spectrum.

吡咯喹啉醌（PQQ）生物合成相关基因簇被确定为pqqABCDEFG。pqqC突变体产生生物合成中间产物，在体外被PqqC蛋白转化为PQQ，并在大肠杆菌中表达和纯化。纯化该中间体，并确认其结构为3a-（2-氨基-2-羧基-乙基）-4，5-二氧代-4，5，6，7，8，9-六氢-醌-7，9-二羧酸。PqqC酶反应需要蛋白因子（ActF）加NADPH以显示完全的酶活性，然而，二硫苏糖醇（DTT）本身可以被替换。当与PQQ混合时，PqqC/D（PqqC和PqqD的融合蛋白）在凝胶过滤柱层析中以复合物形式洗脱，但在DTT存在下彼此分离。在PqqC的情况下，即使在DTT的存在下，部分复合物仍然存在。DTT具有还原PQQ的能力，因此PqqC和PqqC/D可能与氧化的PQQ紧密结合，但与还原形式的PQQ结合不多。通过滴定PQQ测定蛋白质的荧光猝灭，确定PqqC或PqqC/D与PQQ的缔合常数。部分纯化的ActF在PQQ存在下显示NADPH氧化酶活性。提示DTT或ActF加NADPH使PQQ保持还原形式，从而增强PqqC或PqqC/D在体外的PQQ产生。PqqD可能会降低PqqC对还原型PQQ的亲和力。在大肠杆菌中表达PqqE，并在DTT存在下纯化。当DTT缺失时，PqqE似乎更容易被降解。根据其吸收光谱，推测PqqE中含有铁硫原子簇。

项目成果

The structure of a biosynthetic intermediate of pyrroloquinoline quinone (POO) and elucidation of the final step of PQQ biosynthesis.

吡咯喹啉醌 (POO) 生物合成中间体的结构以及 PQQ 生物合成最后一步的阐明。

• 发表时间: 2004
• 期刊: J.Am.Chem.Soc. 126(17)
• 作者: O.T.Magnusson;H.Toyama;et al.
• 通讯作者: et al.

The structure of a biosynthetic intermediate of pyrroloquinoline quinone(POO) and elucidation of the final step of POO biosynthesis.

吡咯喹啉醌（POO）生物合成中间体的结构及POO生物合成最后一步的阐明。

• 发表时间: 2004
• 期刊: J.Am.Chem.Soc. 126(17)
• 作者: O.T.Magnusson;H.Toyama;et al.
• 通讯作者: et al.

Quinate oxidation in Gluconobacter oxydans IF03244 : purification and characterization of quinoprotein quinate dehydrogenase.

氧化葡糖杆菌 IF03244 中的奎宁酸氧化：奎宁蛋白奎宁酸脱氢酶的纯化和表征。

• 发表时间: 2004
• 期刊: FEMS Microbiol.Lett. 241(2)
• 作者: A.S.Vangnai;H.Toyama;et al.
• 通讯作者: et al.

Quinate oxidation in Gluconobacter oxydans IFO3244: purification and characterization of quinoprotein quinate dehydrogenase.

氧化葡糖杆菌 IFO3244 中的奎宁酸氧化：奎宁蛋白奎宁酸脱氢酶的纯化和表征。

• 发表时间: 2004
• 期刊: FEMS Microbiol.Lett. 241(2)
• 作者: A.S.Vangnai;H.Toyama;et al.
• 通讯作者: et al.

Quinone biogenesis: Structure and mechanism of PqqC, the final catalyst in the production of pyrroloquinoline quinone

• DOI: 10.1073/pnas.0402640101
• 发表时间: 2004-05-25
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Magnusson, OT;Toyama, H;Schwarzenbacher, R
• 通讯作者: Schwarzenbacher, R