权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Analysis of novel electron-transporting system in DMSO respiration of a photosynthetic bacterium

光合细菌 DMSO 呼吸中新型电子传输系统的分析

基本信息

批准号：
08456044
负责人：
YAMAZAKI Sunao
金额：
$ 4.74万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08456044/
关键词：
DMSO respiration photodenitrifier dms operon dimethyl sulfoxide tor operon DMSO呼吸 DMSO還元酵素光合成細菌 Rhodobacter sphaeroides dmsC 電子供与活性

项目摘要

A photodenitrifier, Rhodobacter sphaeroides f.sp. denitrifican, can grow under anaerobic-light condition with dimethyl sulfoxide (DMSO) as the terminal electron acceptor in DMSO respiration. The terminal enzyme of this respiration, dimethyl su1foxide redoctase (DMSOR), was recently seqoenced by our team and analyzed crystallographically by U.S.researchers. Attention was focussed worldwide on the relation between roles of molybdenum-cofactor in active site of the enzyme and this novel DMSO respiration system. This research was therefore organized to clarify the details of this respiration system.In the upstream of the gene involving DMSO reductase (dmsA), there was two open reading frames composed of dmsB and cmsC.DmsB was suggested to be low molecular weight membrane protein and dmsC was also membraneous and contains pentaheme with molecular weight of 45000. From the comparison of tor operon in E.coil which has high homology with dms operon, dmsC was deduced as a direct electron-transporter to DMSOR.Many trials were done to extress dmsC in E.coli using varieties of operons, dmsA, dmsB, dmsC, dmsBC, and dmsABC.DmsC was expressed as an inclusion body in the cells but no activity was confirmed. The fact that penetaheme was not introduced in the protein molecule seemed to be the limiting factor to express matured proteins.To elucidate the actual electron flow from dmaC to dmsA, isolation of dmsC from the Rhodobacter cells was performed After many steps of chromatographic purification, dmsC was purified to a single band in SDS-PAGE.Preliminary in-vitro reconstitution experiment, however, showed no activity, indicating that further studies were needed to establish the reconstitution conditions.

光反硝化菌，Rhodobacter sphaeroides f.sp。 denitifican，可以在厌氧光条件下生长，以二甲基亚砜（DMSO）作为DMSO呼吸中的末端电子受体。这种呼吸的末端酶，二甲亚砜还原酶（DMSOR），最近由我们的团队进行了测序，并由美国研究人员进行了晶体学分析。酶活性位点中钼辅因子的作用与这种新型 DMSO 呼吸系统之间的关系引起了全世界的关注。因此，组织本研究来阐明这一呼吸系统的细节。在涉及DMSO还原酶(dmsA)的基因上游，有两个由dmsB和cmsC组成的开放阅读框。DmsB被认为是低分子量膜蛋白，dmsC也是膜蛋白，含有分子量为45000的五血红素。从大肠杆菌中的tor操纵子的比较来看， dmsC 与 dms 操纵子具有高度同源性，因此被推断为 DMSOR 的直接电子转运蛋白。为了在大肠杆菌中表达 dmsC，使用各种操纵子 dmsA、dmsB、dmsC、dmsBC 和 dmsABC 进行了许多试验。DmsC 在细胞中以包涵体形式表达，但未证实其活性。蛋白质分子中未引入 Penetaheme 的事实似乎是表达成熟蛋白质的限制因素。为了阐明从 dmaC 到 dmsA 的实际电子流，从红杆菌细胞中分离 dmsC。经过许多步骤的色谱纯化，dmsC 在 SDS-PAGE 中纯化为单条带。然而，初步的体外重构实验显示没有活性，表明需要进一步研究以确定重组条件。