PhzA/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis
PhzA/B Catalyzes the Formation of the Tricycle in Phenazine Biosynthesis
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DOI：
10.1021/ja806325k
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发表时间：
2008-12-17
影响因子：
15
通讯作者：
Blankenfeldt, Wulf
中科院分区：
文献类型：
--
作者：
Ahuja, Ekta G.;Janning, Petra;Blankenfeldt, Wulf
吩嗪类化合物是具有氧化还原活性的细菌次级代谢产物，参与重要的生物过程，如产生有毒的活性氧和还原环境铁。它们从胆酸的生物合成依赖于phz操纵子编码的酶，但该途径的许多细节仍不清楚。先前表明，吩嗪生物合成涉及两个相同的氨基-环己烯酮分子对称的头-尾双缩合成三环吩嗪前体。虽然这一关键步骤可以在体外自发进行，但我们在这里表明，它是由PhzA/B催化的，PhzA/B是Delta（5）-3-酮甾体异构酶/核转运因子2家族的一种小的二聚体蛋白，我们推断这种催化作用是体内所需的。与底物和产物的类似物复合的晶体结构表明，PhzA/B通过定向两个底物分子和通过质子化中和四面体中间体的负电荷来加速双亚胺的形成。HPLC-耦合NMR显示缩合产物进一步重排，这可能对防止回水解很重要，并且也可能在PhzA/B的活性位点内被催化。重排的三环产物随后在涉及分子氧的非金属依赖性反应中经历氧化脱羧。这种转化似乎不需要酶催化，解释了为什么吩嗪-1-羧酸是一个主要的产品，甚至在菌株中使用吩嗪-1，6-二羧酸作为菌株特异性吩嗪衍生物的前体。
Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.