Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TyIF Family of Sugar O-Methyltransferases.
Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TyIF Family of Sugar O-Methyltransferases.
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DOI：
10.1021/cb5009348
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发表时间：
2015-05-01
影响因子：
4
通讯作者：
Smith, Janet L.
中科院分区：
文献类型：
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作者：
Bernard, Steffen M.;Akey, David L.;Smith, Janet L.
天然产物中的糖部分经常被O-甲基化修饰。在大环内酯类抗生素霉素的生物合成中，6 '-脱氧阿洛糖取代基的甲基化以逐步的方式首先在2'-羟基上发生，然后在3 '-羟基上发生，以在最终产物中产生霉素糖部分。O-甲基化的时机和位置影响由P450单加氧酶MycG介导的最终阶段C-H官能化反应。途径排序和底物特异性的结构基础是未知的。MycF（3 '-O-甲基转移酶）的一系列晶体结构（包括游离酶和与S-腺苷高半胱氨酸（SAH）、底物、产物和非天然底物的复合物）表明，SAM结合诱导了产生天然底物结合位点的实质性有序化，结合的金属离子定位了催化底物。一个单一的氨基酸取代放松了2 '-甲氧基特异性，但保留了区域特异性。工程化的变体产生了一种新的霉素类似物，证明了结构信息的实用性，以促进生物工程方法的化学酶法合成含有修饰的糖的复杂的小分子。使用MycF底物复合物和4 '特异性同源物的模拟底物复合物，鉴定了与MycF家族成员的3'或4'特异性相关的活性位点残基，并定义了指导甲基转移的区域化学的蛋白质和底物特征。这种分类方案将是有用的注释新的次级代谢途径，利用这个家庭的酶。
Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates, show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homologue, active site residues were identified that correlate with the 3' or 4' specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. This classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.