Department of Chemistry, Pomona College, 645 N. College Ave., Claremont, CA 91711, USA.
Arch Biochem Biophys. 2011 Apr 1;508(1):39-45. doi: 10.1016/j.abb.2011.01.010. Epub 2011 Jan 15.
The regiospecific oxidation of aromatic amines to aryl nitro compounds is critical to the synthesis of several natural products having pharmacological importance. The arylamine N-oxygenase (AAO) from Streptomyces thioluteus (AurF) selectively oxidizes p-aminobenzoic acid to p-nitrobenzoic acid and has been the subject of investigation for its unique chemistry and substrate preferences. Little, however, is known about the biochemistry and substrate specificities of AurF homologues, which are often associated with non-ribosomal peptide synthetases or polyketide synthases and have substrate binding pockets with substantially different amino acid compositions based on sequence alignments. An AAO homolog from Pseudomonas syringae pv. phaseolicola was expressed and purified to further explore the substrate specificity and biosynthetic utility of this enzyme class. PsAAO was most active on substituted o-aminophenols at pH 9 in buffer solutions containing 40% methanol. o-Aminophenols allow both the Pseudomonas and Streptomyces AAOs to act on para-substituted arylamines having methoxy, methyl, and nitro groups, which was previously unseen. A Hammett plot of k(cat,app) vs. σ has a ρ = -1.5, indicating substrate reactivity is dependent on the electron donating effects of substituents. The mechanistic data are consistent with an amine lone pair attacking an activated oxygen atom after formation of the hydroperoxy Fe(III/III) intermediate.
芳胺的区域选择性氧化为芳基硝基化合物是合成具有药理重要性的几种天然产物的关键。来自硫链丝菌（AurF）的芳基胺 N-加氧酶（AAO）选择性地将对氨基苯甲酸氧化为对硝基苯甲酸，并且由于其独特的化学性质和底物偏好而成为研究的主题。然而，对于 AurF 同源物的生物化学和底物特异性知之甚少，这些同源物通常与非核糖体肽合成酶或聚酮合酶相关联，并且根据序列比对，其底物结合口袋具有明显不同的氨基酸组成。从菜豆细菌性斑点病菌中表达和纯化了一种 AAO 同源物，以进一步探索该酶类的底物特异性和生物合成用途。在含有 40%甲醇的缓冲溶液中，pH 9 时，PsAAO 在取代的邻氨基酚上的活性最高。邻氨基酚允许假单胞菌和链霉菌 AAO 作用于具有甲氧基、甲基和硝基的对位取代的芳基胺，这是以前未见的。k(cat,app) 与 σ 的 Hammett 图的 ρ = -1.5，表明取代基的电子给体效应决定了底物的反应性。该机理数据与形成过氧 Fe(III/III) 中间物后，胺孤对电子攻击活化氧原子的情况一致。
