Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Biochemistry. 2022 Sep 20;61(18):2025-2035. doi: 10.1021/acs.biochem.2c00350. Epub 2022 Sep 9.
The regiospecific prenylation of an aromatic amino acid catalyzed by a dimethylallyl-l-tryptophan synthase (DMATS) is a key step in the biosynthesis of many fungal and bacterial natural products. DMATS enzymes share a common "ABBA" fold with divergent active site contours that direct alternative C-C, C-N, and C-O bond-forming trajectories. DMATS1 from catalyzes the reverse N-prenylation of l-Trp by generating an allylic carbocation from dimethylallyl diphosphate (DMAPP) that then alkylates the indole nitrogen of l-Trp. DMATS1 stands out among the greater DMATS family because it exhibits unusually broad substrate specificity: it can utilize geranyl diphosphate (GPP) or l-Tyr as an alternative prenyl donor or acceptor, respectively; it can catalyze both forward and reverse prenylation, i.e., at C1 or C3 of DMAPP; and it can catalyze C-N and C-O bond-forming reactions. Here, we report the crystal structures of DMATS1 and its complexes with l-Trp or l-Tyr and unreactive thiolodiphosphate analogues of the prenyl donors DMAPP and GPP. Structures of ternary complexes mimic Michaelis complexes with actual substrates and illuminate active site features that govern prenylation regiochemistry. Comparison with CymD, a bacterial enzyme that catalyzes the reverse N-prenylation of l-Trp with DMAPP, indicates that bacterial and fungal DMATS enzymes share a conserved reaction mechanism. However, the narrower active site contour of CymD enforces narrower substrate specificity. Structure-function relationships established for DMATS enzymes will ultimately inform protein engineering experiments that will broaden the utility of these enzymes as useful tools for synthetic biology.
芳香族氨基酸的区域特异性烯丙基化由二甲基烯丙基-l-色氨酸合酶（DMATS）催化，是许多真菌和细菌天然产物生物合成的关键步骤。DMATS 酶与发散的活性位点轮廓共享共同的“ABBA”折叠，该轮廓指导替代 C-C、C-N 和 C-O 键形成轨迹。来自 的 DMATS1 通过从二甲基烯丙基二磷酸（DMAPP）生成烯丙基碳正离子来催化 l-Trp 的反向 N-烯丙基化，然后该烯丙基碳正离子烷基化 l-Trp 的吲哚氮。DMATS1 在更大的 DMATS 家族中脱颖而出，因为它表现出异常广泛的底物特异性：它可以分别使用香叶基二磷酸（GPP）或 l-Tyr 作为替代烯丙基供体或受体；它可以催化正向和反向烯丙基化，即在 DMAPP 的 C1 或 C3 上；并且可以催化 C-N 和 C-O 键形成反应。在这里，我们报告了 DMATS1 及其与 l-Trp 或 l-Tyr 以及非反应性硫醇二磷酸类似物的复合物的晶体结构，这些类似物分别是烯丙基供体 DMAPP 和 GPP 的前体。三元复合物的结构模拟了具有实际底物的迈克尔复合物，并阐明了控制烯丙基化区域化学的活性位点特征。与催化 l-Trp 与 DMAPP 的反向 N-烯丙基化的细菌酶 CymD 进行比较表明，细菌和真菌 DMATS 酶共享保守的反应机制。然而，CymD 更窄的活性位点轮廓迫使更窄的底物特异性。为 DMATS 酶建立的结构-功能关系最终将为蛋白质工程实验提供信息，这些实验将拓宽这些酶作为合成生物学有用工具的用途。
