Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis.
Initial characterization of Fom3 from Streptomyces wedmorensis: The methyltransferase in fosfomycin biosynthesis.
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DOI：
10.1016/j.abb.2013.12.004
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发表时间：
2014-02-01
影响因子：
3.9
通讯作者：
Wang, Susan C.
中科院分区：
文献类型：
--
作者：
Allen, Kylie D.;Wang, Susan C.
磷霉素是一种广谱抗生素，可有效对抗多重耐药细菌。尽管其生物合成在 40 多年前就已被首次研究，但倒数第二个甲基转移反应的表征一直困扰着研究人员。据信催化该反应的酶 Fom3 已被鉴定为自由基 S-腺苷-L-甲硫氨酸 (SAM) 超家族成员。自由基 SAM 酶使用 SAM 和四铁四硫 ([4Fe-4S]) 簇来催化复杂的化学转化。 Fom3 也属于自由基 SAM 酶家族，该酶包含假定的钴胺素结合基序，表明它使用钴胺素进行甲基化。在这里，我们描述了来自韦德莫伦链霉菌的 Fom3 的第一个生化特征。由于重组 Fom3 不溶，我们开发了成功的重折叠和铁硫簇重建程序。光谱分析表明，Fom3 结合 [4Fe-4S] 簇，该簇经历自由基 SAM 酶特征的 +2“静止”状态和 +1 活性状态之间的转变。自由基 SAM CxxxCxxC 基序中半胱氨酸残基的定点诱变表明每个残基对于功能簇的形成都是必需的。我们还提供了初步证据，表明 Fom3 以明显依赖 SAM、连二亚硫酸钠和甲钴胺的方式将甲基添加到 2-羟乙基膦酸酯 (2-HEP) 上，形成 2-羟丙基膦酸酯 (2-HPP)。
Fosfomycin is a broad-spectrum antibiotic that is useful against multi-drug resistant bacteria. Although its biosynthesis was first studied over 40 years ago, characterization of the penultimate methyl transfer reaction has eluded investigators. The enzyme believed to catalyze this reaction, Fom3, has been identified as a radical S-adenosyl-L-methionine (SAM) superfamily member. Radical SAM enzymes use SAM and a four-iron, four-sulfur ([4Fe-4S]) cluster to catalyze complex chemical transformations. Fom3 also belongs to a family of radical SAM enzymes that contain a putative cobalamin-binding motif, suggesting that it uses cobalamin for methylation. Here we describe the first biochemical characterization of Fom3 from Streptomyces wedmorensis. Since recombinant Fom3 is insoluble, we developed a successful refolding and iron-sulfur cluster reconstitution procedure. Spectroscopic analyses demonstrate that Fom3 binds a [4Fe-4S] cluster which undergoes a transition between a +2 “resting” state and a +1 active state characteristic of radical SAM enzymes. Site-directed mutagenesis of the cysteine residues in the radical SAM CxxxCxxC motif indicates that each residue is essential for functional cluster formation. We also provide preliminary evidence that Fom3 adds a methyl group to 2-hydroxyethylphosphonate (2- HEP) to form 2-hydroxypropylphosphonate (2-HPP) in an apparently SAM-, sodium dithionite-, and methylcobalamin-dependent manner.
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