Divergent evolution of an atypical S-adenosyl-L-methionine-dependent monooxygenase involved in anthracycline biosynthesis
Divergent evolution of an atypical S-adenosyl-L-methionine-dependent monooxygenase involved in anthracycline biosynthesis
复制标题
DOI：
10.1073/pnas.1501765112
复制
发表时间：
2015-08-11
影响因子：
11.1
通讯作者：
Metsa-Ketela, Mikko
中科院分区：
文献类型：
--
作者：
Grocholski, Thadee;Dinis, Pedro;Metsa-Ketela, Mikko
细菌的次级代谢途径负责生物合成数以千计的生物活性天然产物。许多存在于这些途径中的酶已经进化为催化不寻常的化学转化，这是由促进化学多样性的进化压力所促进的。在参与蒽环类抗癌抗生素生物合成的S-腺苷-L-甲硫氨酸（SAM）依赖性甲基转移酶中观察到了这种趋异的酶进化;而来自柔红霉素途径的DnrK是一种典型的4-O-甲基转移酶，来自罗多霉素途径的密切相关的RdmB（52%序列同一性）是一种非典型的10-羟化酶，其需要SAM、硫醇还原剂和分子氧来实现活性。在这里，我们已经使用了广泛的嵌合体深入了解RdmB的功能分化，并表明插入一个单一的丝氨酸残基DnrK是足够的单加氧活性的介绍。DnrK-Ser与阿克拉霉素T和S-腺苷-L-高半胱氨酸复合物的晶体结构精确到1.9埃分辨率，显示插入的丝氨酸S297位于与底物相邻的α-螺旋片段中，但以侧链远离活性位点的方式。进一步的实验工作表明，活性的转变是由前面的苯丙氨酸F296向活性位点的旋转介导的，其阻断了天然DnrK中存在的蛋白质表面的通道。该通道在RdmB中也是封闭的，并且对于在无溶剂环境中的单加氧可能是重要的。最后，我们假设RdmB的羟基化能力源自DnrK的先前未检测到的10-脱羧活性。
Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-L-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-L-homocysteine refined to 1.9-angstrom resolution revealed that the inserted serine S297 resides in an alpha-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.