Andorfer Mary C, Levitz Talya S, Liu Jian, Chakraborty Ankush, King-Roberts Devin T, Nweneka Delight, Imrich Christa N, Drennan Catherine L
Department of Chemistry, Michigan State University, East Lansing, MI 48824.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2025 Aug 12;122(32):e2510389122. doi: 10.1073/pnas.2510389122. Epub 2025 Aug 4.
X-succinate synthases (XSSs) are glycyl radical enzymes (GREs) that catalyze the addition of hydrocarbons to fumarate via radical chemistry, thereby activating them for microbial metabolism. To date, the only structurally characterized XSS is benzylsuccinate synthase (BSS), which functionalizes toluene. A distinct subclass of XSSs acts on saturated hydrocarbons, which possess much stronger C(sp)-H bonds than toluene, suggesting mechanistic and structural differences from BSS. Here, we use cryogenic electron microscopy to determine the structure of one such enzyme, (1-methylalkyl)succinate synthase (MASS) from strain HxN1, which functionalizes -alkanes (C6-C8). The structure reveals an asymmetric dimer in which both sides contain a catalytic α-subunit and accessory γ-subunit. One α-subunit also binds two additional subunits, β and δ. The β-subunit binds a [4Fe-4S] cluster and adopts a fold similar to BSSβ. The β-subunit appears to regulate the flexibility of the α-subunit to enable opening of the active site, affording the binding of -alkane substrates. The δ-subunit, which lacks homology to known GRE subunits, adopts a rubredoxin-like fold that binds a single Fe ion, an architecture not previously reported for GREs. MASSδ occupies the same region of the α-subunit as the activating enzyme (AE) and may regulate the conformational changes required for glycyl radical installation. Structural comparisons between MASS and BSS reveal differences in how fumarate is bound and show amino acid substitutions that could account for the binding of alkanes versus toluene. Together, this structure offers insight into anaerobic alkane activation via fumarate addition.
X-琥珀酸合酶（XSSs）是甘氨酰自由基酶（GREs），通过自由基化学催化将碳氢化合物添加到富马酸酯上，从而使其能够用于微生物代谢。迄今为止，唯一具有结构特征的XSS是使甲苯功能化的苄基琥珀酸合酶（BSS）。XSSs的一个独特亚类作用于饱和烃，饱和烃具有比甲苯更强的C(sp)-H键，这表明其与BSS在机制和结构上存在差异。在这里，我们使用低温电子显微镜来确定一种这样的酶的结构，即来自菌株HxN1的（1-甲基烷基）琥珀酸合酶（MASS），它使正构烷烃（C6-C8）功能化。该结构揭示了一个不对称二聚体，其两侧都包含一个催化α亚基和辅助γ亚基。一个α亚基还结合另外两个亚基，β和δ。β亚基结合一个[4Fe-4S]簇，并采用与BSSβ相似的折叠方式。β亚基似乎调节α亚基的灵活性，以使活性位点打开，从而允许正构烷烃底物的结合。δ亚基与已知的GRE亚基缺乏同源性，采用结合单个铁离子的类红氧还蛋白折叠方式，这是GREs以前未报道过的结构。MASSδ与激活酶（AE）占据α亚基的相同区域，并且可能调节甘氨酰自由基安装所需的构象变化。MASS和BSS之间的结构比较揭示了富马酸酯结合方式的差异，并显示了可能解释烷烃与甲苯结合的氨基酸取代。总之，该结构为通过富马酸酯加成进行厌氧烷烃激活提供了见解。
