Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, Shaanxi, China.
J Am Chem Soc. 2022 Jun 8;144(22):9910-9919. doi: 10.1021/jacs.2c02676. Epub 2022 May 27.
Spectinomycin is a dioxane-bridged, tricyclic aminoglycoside produced by ATCC 27741. While the biosynthetic gene cluster for spectinomycin has been reported, the chemistry underlying construction of the dioxane ring is unknown. The twitch radical SAM enzyme SpeY from the cluster is shown here to catalyze dehydrogenation of the C2' alcohol of (2',3')-tetrahydrospectinomycin to yield (3')-dihydrospectinomycin as a likely biosynthetic intermediate. This reaction is radical-mediated and initiated via H atom abstraction from C2' of the substrate by the 5'-deoxyadenosyl radical equivalent generated upon reductive cleavage of SAM. Crystallographic analysis of the ternary Michaelis complex places serine-183 adjacent to C2' of the bound substrate opposite C5' of SAM. Mutation of this residue to cysteine converts SpeY to the corresponding C2' epimerase mirroring the opposite phenomenon observed in the homologous twitch radical SAM epimerase HygY from the hygromycin B biosynthetic pathway. Phylogenetic analysis suggests a relatively recent evolutionary branching of putative twitch radical SAM epimerases bearing homologous cysteine residues to generate the SpeY clade of enzymes.
壮观霉素是一种由 ATCC 27741 产生的桥连二氧六环三环氨基糖苷。虽然壮观霉素的生物合成基因簇已经被报道，但构建二氧六环的化学原理尚不清楚。来自该簇的 Twitch 自由基 SAM 酶 SpeY 被证明可以催化（2'，3'）-四氢壮观霉素的 C2' 醇的脱氢反应，生成（3'）-二氢壮观霉素作为可能的生物合成中间体。该反应是自由基介导的，通过在 SAM 的还原裂解后生成的 5'-脱氧腺苷自由基等价物从底物的 C2' 上抽取 H 原子而引发。三元 Michaelis 复合物的晶体学分析将丝氨酸-183 置于结合底物的 C2' 旁边，与 SAM 的 C5' 相对。将该残基突变为半胱氨酸会将 SpeY 转化为相应的 C2' 差向异构酶，反映了在 Hygromycin B 生物合成途径中同源 Twitch 自由基 SAM 差向异构酶 HygY 中观察到的相反现象。系统发育分析表明，具有同源半胱氨酸残基的假定 Twitch 自由基 SAM 差向异构酶最近发生了相对进化分支，产生了 SpeY 酶的分支。
