An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway.
An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway.
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DOI：
10.1021/ja511649n
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发表时间：
2015-02-04
影响因子：
15
通讯作者：
Lipscomb, John D.
中科院分区：
文献类型：
--
作者：
Makris, Thomas M.;Vu, Van V.;Meier, Katlyn K.;Komor, Anna J.;Rivard, Brent S.;Muenck, Eckard;Que, Lawrence, Jr.;Lipscomb, John D.
委内瑞拉链霉菌CmlI在非核糖体肽合成酶（NRPS）途径中催化氯霉素的芳胺前体的6电子氧化，产生抗生素的硝基。光学、EPR和Mössbauer研究表明该酶含有非血红素双核铁簇。在团簇的二亚铁态中加入O2会产生寿命特别长的中间体（在4°C时t1/2 = 3小时），根据对18o2敏感的共振拉曼（rR）振动的观察，该中间体被分配为过氧异铁态（cmi -过氧态）。cmli -过氧化物在光谱上不同于非血红素二铁酶的顺式-μ-1,2-过氧化物（μ-η1:η1）中间体。具体来说，它表现出500 nm左右的蓝移宽吸收带和ν(O-O)至少低60 cm−1的rR光谱。Mössbauer过氧态的研究揭示了一个具有小四极分裂和明显的同分异构体位移（0.54和0.62 mm/s）的铁位的不同簇。综上所述，光谱比较清楚地表明cmli -过氧不具有μ-η: 1- η -过氧配体；我们提出μ-η: 1 -η -过氧配体是其独特的光谱性质的原因。cmli -过氧化物与一系列芳胺底物发生二级反应，表明cmli -过氧化物是催化循环的活性物质。从游离芳胺前体高效生产氯霉素表明CmlI催化了生物合成途径的最终步骤，并且前体在此步骤中未与NRPS结合。
Streptomyces venezuelae CmlI catalyzes the 6-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O2 to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t1/2 = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an 18O2-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-μ-1,2-peroxo (μ-η1:η1) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O–O) that is at least 60 cm−1 lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a μ-η1:η1-peroxo ligand; we propose that a μ-η1:η2-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway, and that the precursor is not bound to the NRPS during this step.
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