Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System
Functional Evaluation of the π-Helix in the NAD(P)H:FMN Reductase of the Alkanesulfonate Monooxygenase System
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DOI：
10.1021/acs.biochem.8b00544
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发表时间：
2018-07-31
期刊：
影响因子：
2.9
通讯作者：
Ellis, Holly R.
中科院分区：
文献类型：
--
作者：
Musila, Jonathan M.;Forbes, Dianna L.;Ellis, Holly R.
NAD（P）H：FMN还原酶家族中的酶亚组由来自双组分单加氧酶系统的黄素还原酶组成。这些FMN还原酶中的发散结构特征是位于四聚体界面中心的π-螺旋，其通过在保守的α 4螺旋中插入氨基酸而产生。SsuE的Tyr插入残基在二聚体界面上产生特异性接触，这可能有助于改变该酶的机械性质。Y118 F SsuE变体在四聚体界面处保持π-π堆积相互作用，并且具有与野生型SsuE相似的动力学参数。取代的π-螺旋残基（Tyr 118）丙氨酸或丝氨酸转化成黄素结合的SsuE变异体，不能再支持黄素还原酶和糖化活性的酶。这些变体以二聚体形式存在，即使黄素转移不持续，也可以与SsuD形成蛋白质-蛋白质相互作用。Delta Y118 SsuE变体在纯化时是无黄素的，并且在添加黄素的情况下不经历四聚体到二聚体的寡聚体转变。缺乏黄素化活性可归因于Delta Y118 SsuE不能促进黄素转移并经历支持黄素化所需的寡聚体变化。这些研究的结果提供了深入了解SsuE π-螺旋在促进黄素转移和寡聚体变化中的作用，这些变化支持蛋白质与SsuD的相互作用。
A subgroup of enzymes in the NAD(P)H:FMN reductase family is comprised of flavin reductases from two-component monooxygenase systems. The diverging structural feature in these FMN reductases is a pi-helix centrally located at the tetramer interface that is generated by the insertion of an amino acid in a conserved alpha 4 helix. The Tyr insertional residue of SsuE makes specific contacts across the dimer interface that may assist in the altered mechanistic properties of this enzyme. The Y118F SsuE variant maintained the pi-pi stacking interactions at the tetramer interface and had kinetic parameters similar to those of wild-type SsuE. Substitution of the pi-helical residue (Tyr118) to Ala or Ser transformed the enzymes into flavin-bound SsuE variants that could no longer support flavin reductase and desulfonation activities. These variants existed as dimers and could form protein protein interactions with SsuD even though flavin transfer was not sustained. The Delta Y118 SsuE variant was flavin-free as purified and did not undergo the tetramer to dimer oligomeric shift with the addition of flavin. The absence of desulfonation activity can be attributed to the inability of Delta Y118 SsuE to promote flavin transfer and undergo the requisite oligomeric changes to support desulfonation. Results from these studies provide insights into the role of the SsuE pi-helix in promoting flavin transfer and oligomeric changes that support protein protein interactions with SsuD.