A Four-Enzyme Pathway for 3,5-Dihydroxy-4-methylanthranilic Acid Formation and Incorporation into the Antitumor Antibiotic Sibiromycin
A Four-Enzyme Pathway for 3,5-Dihydroxy-4-methylanthranilic Acid Formation and Incorporation into the Antitumor Antibiotic Sibiromycin
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DOI：
10.1021/bi2006114
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发表时间：
2011-06-28
期刊：
影响因子：
2.9
通讯作者：
Marahiel, Mohamed A.
中科院分区：
文献类型：
--
作者：
Giessen, Tobias W.;Kraas, Femke I.;Marahiel, Mohamed A.
抗肿瘤抗生素西伯霉素属于吡咯并[1,4]苯二氮卓类 (PBD) 类，由多种放线菌产生。 PBD 是序列特异性 DNA 烷化剂，具有显着的抗肿瘤特性。其中，西伯霉素是已鉴定的两种糖基化 PBD 之一，具有最高的 DNA 结合亲和力和最有效的抗肿瘤活性。在这项研究中，我们从已知的代谢物 3-羟基犬尿氨酸 (3HK) 开始，阐明了西伯霉素中发现的 3,5-二羟基-4-甲基邻氨基苯酸结构单元的形成和激活的精确反应序列。所研究的途径由四种酶组成，并在体外进行了生化表征。从 3HK 开始，SAM 依赖性甲基转移酶 SibL 将底物转化为其 4-甲基衍生物，然后通过 PLP 依赖性犬尿氨酸酶 SibQ 的作用进行水解，形成 3-羟基-4-甲基邻氨基苯甲酸 (3H4MAA)。随后，NRPS 双结构域 SibE 激活 3H4MAA 并将其束缚到其硫醇化结构域，在该结构域中，它在 C5 位置被 FAD/NADH 依赖性羟化酶 SibG 羟基化，产生西伯霉素中发现的完全取代的邻氨基苯甲酸部分。这些关于西伯霉素生物合成和所涉及的生物合成酶的底物特异性的见解可能会指导未来设计 PBD 生物合成机制的尝试，并有助于 PBD 衍生物的生产。
The antitumor antibiotic sibiromycin belongs to the class of pyrrolo[1,4]benzodiazepines (PBDs) that are produced by a variety of actinomycetes. PBDs are sequence-specific DNA-alkylating agents and possess significant antitumor properties. Among them, sibiromycin, one of two identified glycosylated PBDs, displays the highest DNA binding affinity and the most potent antitumor activity. In this study, we report the elucidation of the precise reaction sequence leading to the formation and activation of the 3,5-dihydroxy-4-methylanthranilic acid building block found in sibiromycin, starting from the known metabolite 3-hydroxykynurenine (3HK). The investigated pathway consists of four enzymes, which were biochemically characterized in vitro. Starting from 3HK, the SAM-dependent methyltransferase SibL converts the substrate to its 4-methyl derivative, followed by hydrolysis through the action of the PLP-dependent kynureninase SibQ, leading to 3-hydroxy-4-methylanthranilic acid (3H4MAA) formation. Subsequently the NRPS didomain SibE activates 3H4MAA and tethers it to its thiolation domain, where it is hydroxylated at the C5 position by the FAD/NADH-dependent hydroxylase SibG yielding the fully substituted anthranilate moiety found in sibiromycin. These insights about sibiromycin biosynthesis and the substrate specificities of the biosynthetic enzymes involved may guide future attempts to engineer the PBD biosynthetic machinery and help in the production of PBD derivatives.