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

Elucidation of molecular mechanism of bilin synthesis by ferredoxin-dependent bilin reductases

阐明铁氧还蛋白依赖性胆素还原酶合成胆素的分子机制

基本信息

批准号：
20370037
负责人：
FUKUYAMA Keiichi
金额：
$ 12.81万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2010
项目状态：
已结题

项目摘要

In order to elucidate the molecular mechanism of PcyA, we determined the crystal structures of PcyA in complex with the reaction intermediate (18^1,18^2-dihydrobiliverdin), PcyA in complex with the synthetic analog of substrate (biliverdin XIIIα), and PcyA mutant protein of E76Q in complex with BV. These structural studies showed that the carboxyl group of Glu76 in PcyA-BV was in close proximity to the D-ring vinyl group, suggesting the presence of OH...π hydrogen bond here and that this bond is a key for the preceding reduction of the D-ring vinyl group.When Val225 was replaced by Asp, the absorption spectrum of this mutant protein in complex with BV was very different from that of PcyA-BV. The orientation of BV in V225D-BV was inverted and the induced-fit did not occur upon BV binding. This observation presented a cautionary note about interpreting functional data derived from a mutated protein in the absence of its exact structure. In parallel we expressed, purified and crystallized cyanobacterial biliverdin reductase, an enzyme that reduces at different site of BV from PcyA, We also prepared and crystallized the Se-Met form of biliverdin reductase, and performed preliminary X-ray diffraction analyses for these crystals.We determined the crystal structure of red chlorophyll catabolite reductase (RCCR), a key enzyme in the chlorophyll breakdown pathway. The structure was essentially the same as PcyA, implicating that RCCR belongs to the ferredoxin-dependent bilin reductase family. We also determined the crystal structures of RCCR and its mutant protein in complex with substrate, proposing the reaction mechanism as well as the active residues of RCCR.

为了阐明PcyA的分子机制，我们测定了PcyA与反应中间体(18^1,18^2-二氢胆绿素)复合物、PcyA与底物合成类似物(胆绿素XIIIα)复合物以及PcyA与BV复合物的E76Q突变蛋白的晶体结构。这些结构研究表明，PcyA-BV中Glu76的羧基与D环乙烯基非常接近，表明这里存在OH...π氢键，并且该键是之前D环乙烯基还原的关键。当Val225被Asp取代时，与BV复合的该突变蛋白的吸收光谱与PcyA-BV有很大不同。 V225D-BV 中 BV 的方向是反向的，并且 BV 结合时没有发生诱导拟合。这一观察结果对在缺乏确切结构的情况下解释源自突变蛋白的功能数据提出了警告。与此同时，我们表达、纯化和结晶了蓝藻胆绿素还原酶，这是一种在 BV 的不同位点还原 PcyA 的酶。我们还制备并结晶了 Se-Met 形式的胆绿素还原酶，并对这些晶体进行了初步的 X 射线衍射分析。我们确定了红色叶绿素分解代谢物还原酶 (RCCR) 的晶体结构，这是一个关键的酶。叶绿素分解途径中的酶。其结构与 PcyA 基本相同，表明 RCCR 属于铁氧还蛋白依赖性胆碱还原酶家族。我们还确定了RCCR及其突变蛋白与底物复合物的晶体结构，提出了反应机理以及RCCR的活性残基。