A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.
A Priming Cassette Generates Hydroxylated Acyl Starter Units in Mupirocin and Thiomarinol Biosynthesis.
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引发盒在莫匹罗星和硫代马林醇生物合成中生成羟基化酰基起始单元。
DOI：
10.1021/acschembio.9b00969
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发表时间：
2020
影响因子：
4
通讯作者：
Walker PD
中科院分区：
文献类型：
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作者：
Walker PD
莫匹罗星（一种市售抗生素，由荧光假单胞菌 NCIMB 10586 生产）和硫代马林醇（从海洋细菌假交替单胞菌属 sp. 中分离出来）。 SANK 73390 均由聚酮化合物衍生的单酸同系物组成，经 9-羟基壬酸（莫匹罗星，9HN）或 8-羟基辛酸（thiomarinol，8HO）酯化。这些看似简单的 9HN 和 8HO 脂肪酸部分分别在 mup 和 tml 中形成的机制仍未解决。为了定义起始单元的生成，纯化的莫匹罗星蛋白 MupQ、MupS 和 MacpD 及其 thiomarinol 等效物（TmlQ、TmlS 和 TacpD）已被表达，并显示可通过MupQ/TmlQ 催化生成一种不寻常的双辅酶 A/酰基载体蛋白 (ACP) 硫酯，然后 MupS/TmlS 催化还原。混合搭配实验表明 MupQ/TmlQ 对正确的 CoA 具有高度选择性。 MacpD/TacpD 可以互换，但来自莫匹罗星途径的替代反式作用 ACP (MacpA/TacpA) 或异源 ACP (BatA) 没有功能。 MupS 和 TmlS 的选择性更加多样化，并且这些还原酶的底物和 ACP 选择性有所不同。通过 NMR 确定的 MacpD 溶液结构揭示了具有部分螺旋特征的 C 末端延伸，这已被证明对于维持莫匹罗星的高滴度非常重要。我们生成了截短的 MacpD 构建体 MacpD_T，它缺少此 C 末端延伸，但保留了与 MupS 和 MupQ 生成 3-HP 的能力，表明 ACP 该区域在蛋白质-蛋白质相互作用中具有进一步的下游作用。
Mupirocin, a commercially available antibiotic produced byPseudomonas fluorescensNCIMB 10586, and thiomarinol, isolated from the marine bacteriumPseudoalteromonassp. SANK 73390, both consist of a polyketide-derived monic acid homologue esterified with either 9-hydroxynonanoic acid (mupirocin, 9HN) or 8-hydroxyoctanoic acid (thiomarinol, 8HO). The mechanisms of formation of these deceptively simple 9HN and 8HO fatty acid moieties inmupandtml, respectively, remain unresolved. To define starter unit generation, the purified mupirocin proteins MupQ, MupS, and MacpD and their thiomarinol equivalents (TmlQ, TmlS and TacpD) have been expressed and shown to convert malonyl coenzyme A (CoA) and succinyl CoA to 3-hydroxypropionoyl (3-HP) or 4-hydroxybutyryl (4-HB) fatty acid starter units, respectively,viathe MupQ/TmlQ catalyzed generation of an unusual bis-CoA/acyl carrier protein (ACP) thioester, followed by MupS/TmlS catalyzed reduction. Mix and match experiments show MupQ/TmlQ to be highly selective for the correct CoA. MacpD/TacpD were interchangeable but alternatetrans-acting ACPs from the mupirocin pathway (MacpA/TacpA) or a heterologous ACP (BatA) were nonfunctional. MupS and TmlS selectivity was more varied, and these reductases differed in their substrate and ACP selectivity. The solution structure of MacpD determined by NMR revealed a C-terminal extension with partial helical character that has been shown to be important for maintaining high titers of mupirocin. We generated a truncated MacpD construct, MacpD_T, which lacks this C-terminal extension but retains an ability to generate 3-HP with MupS and MupQ, suggesting further downstream roles in protein–protein interactions for this region of the ACP.