Miyake Keita, Iwata Saya, Narikawa Rei
Department of General Systems Studies, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro, Tokyo, 153-8902 Japan.
Department of Biological Science, Faculty of Science, Shizuoka University, 836 Ohya, Sumga-ku, Shizuoka, 422-8529 Japan.
Plant Cell Physiol. 2025 Feb 28;66(2):229-237. doi: 10.1093/pcp/pcae098.
Phycourobilin:ferredoxin oxidoreductase (PubS) belongs to the ferredoxin-dependent bilin reductase (FDBR) family and catalyzes the reduction of the C15=C16 double bond, followed by the C4=C5 double bond of biliverdin IXα to produce phycourobilin. Among the diverse FDBR enzymes that catalyze site-specific reduction reactions of bilins, PubS lineage is the only one that reduces the C4=C5 double bond. This family can be broadly divided into four-electron reduction enzymes, which catalyze two successive two-electron reductions, such as PubS, and two-electron reduction enzymes, which catalyze a single two-electron reduction. The crystal structures of diverse FDBRs, excluding PubS, have unraveled that there are two distinct binding modes in the substrate-binding pocket. In this study, we focused on the arginine (Arg) residues that is considered crucial for substrate-binding mode in two-electron reduction enzymes. Through sequence alignment and comparison with the predicted structure of PubS, we identified a residue in PubS that corresponds to the Arg residue in the two-electron reduction enzymes. We further introduced mutations to avoid the steric hindrance associated with changes in the binding mode. Biochemical characterization of these variants showed that we successfully modified PubS from a four-electron reduction enzyme to a two-electron reduction enzyme with the accumulation of radicals. Our results provide insight into the molecular mechanisms of the chromophore binding mode and proton donation from acidic residues.
铁氧化还原蛋白氧化还原酶（PubS）属于依赖铁氧化还原蛋白的胆素还原酶（FDBR）家族，催化胆红素原IXα的C15 = C16双键还原，随后是C4 = C5双键还原，生成藻尿胆素。在催化胆素位点特异性还原反应的多种FDBR酶中，PubS谱系是唯一能还原C4 = C5双键的。该家族可大致分为催化两次连续双电子还原的四电子还原酶（如PubS）和催化单次双电子还原的双电子还原酶。除PubS外，多种FDBR的晶体结构揭示，底物结合口袋中有两种不同的结合模式。在本研究中，我们聚焦于双电子还原酶中对底物结合模式至关重要的精氨酸（Arg）残基。通过序列比对并与PubS的预测结构比较，我们在PubS中鉴定出一个与双电子还原酶中的Arg残基对应的残基。我们进一步引入突变以避免与结合模式变化相关的空间位阻。这些变体的生化特性表明，我们成功地将PubS从一种四电子还原酶修饰为一种有自由基积累的双电子还原酶。我们的结果为发色团结合模式和酸性残基质子供体的分子机制提供了见解。
