Department of Chemistry and Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2013 May 21;110(21):8519-24. doi: 10.1073/pnas.1302417110. Epub 2013 May 6.
Arylsulfatases require a maturating enzyme to perform a co- or posttranslational modification to form a catalytically essential formylglycine (FGly) residue. In organisms that live aerobically, molecular oxygen is used enzymatically to oxidize cysteine to FGly. Under anaerobic conditions, S-adenosylmethionine (AdoMet) radical chemistry is used. Here we present the structures of an anaerobic sulfatase maturating enzyme (anSME), both with and without peptidyl-substrates, at 1.6-1.8 Å resolution. We find that anSMEs differ from their aerobic counterparts in using backbone-based hydrogen-bonding patterns to interact with their peptidyl-substrates, leading to decreased sequence specificity. These anSME structures from Clostridium perfringens are also the first of an AdoMet radical enzyme that performs dehydrogenase chemistry. Together with accompanying mutagenesis data, a mechanistic proposal is put forth for how AdoMet radical chemistry is coopted to perform a dehydrogenation reaction. In the oxidation of cysteine or serine to FGly by anSME, we identify D277 and an auxiliary [4Fe-4S] cluster as the likely acceptor of the final proton and electron, respectively. D277 and both auxiliary clusters are housed in a cysteine-rich C-terminal domain, termed SPASM domain, that contains homology to ~1,400 other unique AdoMet radical enzymes proposed to use [4Fe-4S] clusters to ligate peptidyl-substrates for subsequent modification. In contrast to this proposal, we find that neither auxiliary cluster in anSME bind substrate, and both are fully ligated by cysteine residues. Instead, our structural data suggest that the placement of these auxiliary clusters creates a conduit for electrons to travel from the buried substrate to the protein surface.
芳基硫酸酯酶需要一种成熟酶来进行共翻译或翻译后修饰，以形成催化必需的甲酰甘氨酸 (FGly) 残基。在需氧生物中，分子氧被酶促氧化半胱氨酸形成 FGly。在厌氧条件下，使用 S-腺苷甲硫氨酸 (AdoMet) 自由基化学。在这里，我们展示了 1.6-1.8 Å 分辨率的具有和不具有肽基底物的厌氧硫酸酯酶成熟酶 (anSME) 的结构。我们发现 anSME 与需氧对应物的不同之处在于，它们使用基于骨架的氢键模式与肽基底物相互作用，从而降低了序列特异性。这些来自产气荚膜梭菌的 anSME 结构也是第一个进行脱氢化学的使用 AdoMet 自由基酶的结构。结合伴随的突变体数据，提出了一种关于如何利用 AdoMet 自由基化学进行脱氢反应的机制建议。在 anSME 将半胱氨酸或丝氨酸氧化为 FGly 的过程中，我们确定 D277 和一个辅助 [4Fe-4S] 簇分别是最终质子和电子的可能受体。D277 和两个辅助簇都位于富含半胱氨酸的 C 末端结构域中，称为 SPASM 结构域，该结构域与 ~1400 个其他独特的 AdoMet 自由基酶具有同源性，这些酶被认为使用 [4Fe-4S] 簇来连接肽基底物以进行随后的修饰。与该提议相反，我们发现 anSME 中的两个辅助簇都不结合底物，并且都完全由半胱氨酸残基连接。相反，我们的结构数据表明，这些辅助簇的位置为电子从埋藏的底物到蛋白质表面的传输创建了一个通道。
