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

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)的络合物、PcyA与合成底物类似物(胆绿素XIIIα)的络合物以及PcyA突变蛋白E76Q与BV的络合物的晶体结构。这些结构研究表明，PcyA-BV中Glu76的羧基与D-环乙烯基的位置很接近，这表明在PcyA-BV中存在OH…π氢键，这一键是D-环乙烯基还原的关键。当用天冬氨酸取代Val225时，该突变蛋白与BV形成的复合物的吸收光谱与PcyA-BV有很大不同。V225D-BV中的BV取向颠倒，BV结合后没有发生诱导匹配。这一观察对在缺乏确切结构的情况下解释来自突变蛋白质的功能数据提出了警告。同时，我们对蓝藻胆绿素还原酶进行了表达、纯化和结晶，制备并结晶了胆绿素还原酶的Se-Met形式，并对其进行了X射线衍射分析，确定了叶绿素降解途径中的关键酶--红色叶绿素分解代谢还原酶(RCCR)的晶体结构。其结构与PcyA基本相同，提示RCCR属于铁氧还蛋白依赖的胆碱还原酶家族。我们还测定了RCCR及其突变蛋白在底物络合物中的晶体结构，提出了RCCR的反应机理和活性残基。

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.