Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N⁶-acetyl-N⁶-hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis.

中文翻译：

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航空细菌素生物合成途径的铁载体合成酶IucA使用一种有序的机制。

异羟肟酸酯铁氧体航空细菌素的生物合成需要iuc操纵子内编码的四种蛋白质的活性。最近，我们通过生物化学方法重建了生物合成途径，并在结构上表征了依次连接N ^6-乙酰-N ^6的两种酶IucA和IucC。-羟基赖氨酸生成柠檬酸的伯羧酸盐。IucA和IucC是不依赖核糖体肽合成酶的铁载体（NIS）合成酶家族的成员，该酶与其他铁载体的生产有关，包括去铁草胺，脱色杆菌素和petrobactin。虽然该家族的几个成员的结构先前已得到解决，但对由NIS合成酶催化的反应的机理了解有限。因此，我们进行了三级反应物稳态动力学分析，并在此为有序机制提供了证据，其中化学反应之前形成了季铵盐配合物。我们通过定点诱变进一步探测了活性位点的两个区域，并鉴定了几个残基，包括存在于动态环上的保守基序，