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

光合细菌Rhodobacter sphaeroides f.sp. pestican能在厌氧光照条件下生长，以二甲基亚砜（DMSO）作为DMSO呼吸的末端电子受体。这种呼吸作用的末端酶，二甲基亚砜还原酶（DMSOR），最近由我们的团队测序，并由美国研究人员进行晶体学分析。酶活性中心的辅酶与这种新型DMSO呼吸系统之间的关系引起了全世界的关注。在DMSA基因的上游有两个开放的阅读框，分别由DMSB和CMSC组成，DMSB是低分子量的膜蛋白，DMSC也是膜蛋白，含有分子量为45000的五血红素。通过比较大肠杆菌中tor操纵子与dms操纵子的同源性，推测dmsC是DMSOR的直接电子转运体，并利用dmsA、dmsB、dmsC、dmsBC和dmsABC等多种操纵子对大肠杆菌中的dmsC进行了大量的抽提实验，结果表明dmsC在大肠杆菌中以包涵体形式表达，但没有活性。为了阐明dmaC到dmsA的电子流，从红细菌细胞中分离了dmsC，经多次层析纯化后，dmsC在SDS-PAGE中被纯化为单一条带，但初步的体外重组实验显示没有活性，这表明需要进一步的研究来建立重构条件。

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.