PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa
PqsL uses reduced flavin to produce 2-hydroxylaminobenzoylacetate, a preferred PqsBC substrate in alkyl quinolone biosynthesis in Pseudomonas aeruginosa
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DOI：
10.1074/jbc.ra117.000789
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发表时间：
2018-06-15
影响因子：
4.8
通讯作者：
Fetzner, Susanne
中科院分区：
文献类型：
--
作者：
Drees, Steffen Lorenz;Ernst, Simon;Fetzner, Susanne
烷基羟基喹啉 N-氧化物 (AQNO) 是由机会性细菌病原体铜绿假单胞菌产生的抗生素化合物。它们是烷基喹诺酮 (AQ) 生物合成途径的产物，该途径还生成群体感应分子 2-庚基-4(1H)-喹诺酮 (HHQ) 和 2-庚基-3-羟基-4(1H)-喹诺酮 (PQS)。尽管 HHQ 和 PQS 的酶促合成已被阐明，但 AQNO 的合成途径仍不清楚。在此，我们报道了 PqsL，它是 AQNO 生产的关键酶，其结构类似于 A 类黄素蛋白单加氧酶，如对羟基苯甲酸 3-羟化酶 (pHBH) 和 3-羟基苯甲酸 6-羟化酶。然而，我们发现，与相关酶不同，PqsL 羟基化伯芳香胺基团，并且它不使用 NAD(P)H 作为共底物，但出乎意料地需要还原黄素作为电子供体。我们还观察到 PqsL 对 AQ 途径的中心中间体 2-氨基苯甲酰乙酸 (2-ABA) 具有活性，并形成不稳定的化合物 2-羟氨基苯甲酰乙酸，它比 2-ABA 更适合作为下游酶 PqsBC 的底物。通过使用 FAD 还原酶 HpaC 可以体外重建 PqsL/PqsBC 反应，并且我们注意到与 WT 菌株中的比率相比，铜绿假单胞菌 PAO1 的 hpaC 缺失突变体中的 AQ:AQNO 比率有所增加。与 A 类黄素蛋白单加氧酶的模型酶 pHBH 的结构比较表明，PqsL 中缺少与 NAD(P)H 结合相关的结构特征。我们的研究完成了铜绿假单胞菌中的 AQNO 生物合成途径，表明 PqsL 从 2-ABA 产生不稳定的产物 2-羟氨基苯甲酰乙酸，并依赖于游离还原黄素作为电子供体，而不是 NAD(P)H。
Alkyl hydroxyquinoline N-oxides (AQNOs) are antibiotic compounds produced by the opportunistic bacterial pathogen Pseudomonas aeruginosa. They are products of the alkyl quinolone (AQ) biosynthetic pathway, which also generates the quorum-sensing molecules 2-heptyl-4(1H)-quinolone (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS). Although the enzymatic synthesis of HHQ and PQS had been elucidated, the route by which AQNOs are synthesized remained elusive. Here, we report on PqsL, the key enzyme for AQNO production, which structurally resembles class A flavoprotein monooxygenases such as p-hydroxybenzoate 3-hydroxylase (pHBH) and 3-hydroxybenzoate 6-hydroxylase. However, we found that unlike related enzymes, PqsL hydroxylates a primary aromatic amine group, and it does not use NAD(P)H as cosubstrate, but unexpectedly required reduced flavin as electron donor. We also observed that PqsL is active toward 2-aminobenzoylacetate (2-ABA), the central intermediate of the AQ pathway, and forms the unstable compound 2-hydroxylaminobenzoylacetate, which was preferred over 2-ABA as substrate of the downstream enzyme PqsBC. In vitro reconstitution of the PqsL/PqsBC reaction was feasible by using the FAD reductase HpaC, and we noted that the AQ:AQNO ratio is increased in an hpaC-deletion mutant of P. aeruginosa PAO1 compared with the ratio in the WT strain. A structural comparison with pHBH, the model enzyme of class A flavoprotein monooxygenases, revealed that structural features associated with NAD(P)H binding are missing in PqsL. Our study completes the AQNO biosynthetic pathway in P. aeruginosa, indicating that PqsL produces the unstable product 2-hydroxylaminobenzoylacetate from 2-ABA and depends on free reduced flavin as electron donor instead of NAD(P)H.