Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA.
Department of Chemistry, Boston University, Boston, MA 02215, USA.
Cell Chem Biol. 2018 May 17;25(5):519-529.e4. doi: 10.1016/j.chembiol.2018.02.002. Epub 2018 Mar 1.
Sulfur incorporation in the biosynthesis of ergothioneine, a histidine thiol derivative, differs from other well-characterized transsulfurations. A combination of a mononuclear non-heme iron enzyme-catalyzed oxidative C-S bond formation and a subsequent pyridoxal 5'-phosphate (PLP)-mediated C-S lyase reaction leads to the net transfer of a sulfur atom from a cysteine to a histidine. In this study, we structurally and mechanistically characterized a PLP-dependent C-S lyase Egt2, which mediates the sulfoxide C-S bond cleavage in ergothioneine biosynthesis. A cation-π interaction between substrate and enzyme accounts for Egt2's preference of sulfoxide over thioether as a substrate. Using mutagenesis and structural biology, we captured three distinct states of the Egt2 C-S lyase reaction cycle, including a labile sulfenic intermediate captured in Egt2 crystals. Chemical trapping and high-resolution mass spectrometry were used to confirm the involvement of the sulfenic acid intermediate in Egt2 catalysis.
硫掺入到麦硫因的生物合成中，这是一种组氨酸硫醇衍生物，与其他特征明确的转硫作用不同。一个单核非血红素铁酶催化的氧化 C-S 键形成，以及随后的吡哆醛 5′-磷酸（PLP）介导的 C-S 裂合酶反应的组合，导致一个硫原子从半胱氨酸净转移到组氨酸。在这项研究中，我们从结构和机制上对依赖 PLP 的 C-S 裂合酶 Egt2 进行了表征，该酶介导麦硫因生物合成中硫氧化物的 C-S 键断裂。底物与酶之间的阳离子-π 相互作用解释了 Egt2 偏爱作为底物的硫氧化物而不是硫醚的原因。通过突变和结构生物学，我们捕获了 Egt2 C-S 裂合酶反应循环的三个不同状态，包括在 Egt2 晶体中捕获的不稳定亚磺酸中间产物。化学捕获和高分辨率质谱用于确认亚磺酸中间物在 Egt2 催化中的参与。
