Biochemical and Structural Characterization of TtnD, a Prenylated FMN-Dependent Decarboxylase from the Tautomycetin Biosynthetic Pathway
Biochemical and Structural Characterization of TtnD, a Prenylated FMN-Dependent Decarboxylase from the Tautomycetin Biosynthetic Pathway
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DOI：
10.1021/acschembio.8b00673
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发表时间：
2018-09-01
影响因子：
4
通讯作者：
Shen, Ben
中科院分区：
文献类型：
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作者：
Annaval, Thibault;Han, Lu;Shen, Ben
互变霉素（TTN）是一种末端为烯烃的聚酮化合物天然产物。来自灰色产色链霉菌的ttn基因簇内的基因的功能表征建立了TTN聚酮骨架的生物合成、其二烷基马来酸酐部分、两个部分的偶联以形成新生中间体TTN F-1以及将TTN F-1转化为TTN的剪裁步骤。在这里，我们报告的生物化学和结构表征TtnD，异戊二烯化FMN（prFMN）依赖性脱羧酶属于UbiD家族，催化TTN生物合成的倒数第二步。TtnD催化TTN D-1脱羧为TTN I-1，利用prFMN作为由TtnC黄素异戊烯基转移酶产生的辅因子; TtnD和TtnC均在ttn生物合成基因簇内编码。TtnD表现出底物混杂性，但仅接受以α，α/β-不饱和酸为特征的TTN D-1同源物，从而在需要α，α/β-不饱和酸底物的双键的催化过程中支持[3+2]环加成机制。TtnD与UbiD家族的其他成员共享类似的整体结构，但在溶液中形成同源四聚体。每个原聚体由三个结构域组成，其中活性位点位于中间和C-末端结构域之间;通过定点诱变证实了R169 E272-E277（构成催化三联体）和E228（参与Mn（II）介导的prFMN结合）。TtnD代表参与聚酮生物合成的prFMN依赖性脱羧酶的第一个实例，将脱羧酶的UbiD家族的底物范围扩展到简单的芳香酸和肉桂酸之外。TtnD及其同系物广泛存在于自然界中，可用作有机合成的生物催化剂。
Tautomycetin (TTN) is a polyketide natural product featuring a terminal alkene. Functional characterization of the genes within the ttn gene cluster from Streptomyces griseochromogenes established the biosynthesis of the TTN polyketide backbone, its dialkylmaleic anhydride moiety, the coupling of the two moieties to form the nascent intermediate TTN F-1, and the tailoring steps converting TTN F-1 to TTN. Here, we report biochemical and structural characterization of TtnD, a prenylated FMN (prFMN)-dependent decarboxylase belonging to the UbiD family that catalyzes the penultimate step of TTN biosynthesis. TtnD catalyzes decarboxylation of TTN D-1 to TTN I-1, utilizing prFMN as a cofactor generated by the TtnC flavin prenyltransferase; both TtnD and TtnC are encoded within the ttn biosynthetic gene cluster. TtnD exhibits substrate promiscuity but accepts only TTN D-1 congeners that feature an a,alpha/beta-unsaturated acid, supporting the [3+2] cycloaddition mechanism during catalysis that requires the double bond of an a,alpha/beta-unsaturated acid substrate. TtnD shares a similar overall structure with other members of the UbiD family but forms a homotetramer in solution. Each protomer is composed of three domains with the active site located between the middle and C-terminal domains; R169 E272-E277, constituting the catalytic triad, and E228, involved in Mn(II)-mediated binding of prFMN, were confirmed by site directed mutagenesis. TtnD represents the first example of a prFMN-dependent decarboxylase involved in polyketide biosynthesis, expanding the substrate scope of the UbiD family of decarboxylases beyond simple aromatic and cinnamic acids. TtnD and its homologues are widespread in nature and could be exploited as biocatalysts for organic synthesis.