Liu Pinghua, Liu Aimin, Yan Feng, Wolfe Matt D, Lipscomb John D, Liu Hung-wen
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas, Austin, Texas 78712, USA.
Biochemistry. 2003 Oct 14;42(40):11577-86. doi: 10.1021/bi030140w.
The last step of the biosynthesis of fosfomycin, a clinically useful antibiotic, is the conversion of (S)-2-hydroxypropylphosphonic acid (HPP) to fosfomycin. Since the ring oxygen in fosfomycin has been shown in earlier feeding experiments to be derived from the hydroxyl group of HPP, this oxirane formation reaction is effectively a dehydrogenation process. To study this unique C-O bond formation step, we have overexpressed and purified the desired HPP epoxidase. Results reported herein provided initial biochemical evidence revealing that HPP epoxidase is an iron-dependent enzyme and that both NAD(P)H and a flavin or flavoprotein reductase are required for its activity. The 2 K EPR spectrum of oxidized iron-reconstituted fosfomycin epoxidase reveals resonances typical of S = (5)/(2) Fe(III) centers in at least two environments. Addition of HPP causes a redistribution with the appearance of at least two additional species, showing that the iron environment is perturbed. Exposure of this sample to NO elicits no changes, showing that the iron is nearly all in the Fe(III) state. However, addition of NO to the Fe(II) reconstituted enzyme that has not been exposed to O(2) yields an intense EPR spectrum typical of an S = (3)/(2) Fe(II)-NO complex. This complex is also heterogeneous, but addition of substrate converts it to a single, homogeneous S = (3)/(2) species with a new EPR spectrum, suggesting that substrate binds to or near the iron, thereby organizing the center. The fact that NO binds to the ferrous center suggests O(2) can also bind at this site as part of the catalytic cycle. Using purified epoxidase and (18)O isotopic labeled HPP, the retention of the hydroxyl oxygen of HPP in fosfomycin was demonstrated. While ether ring formation as a result of dehydrogenation of a secondary alcohol has precedence in the literature, these catalyses require alpha-ketoglutarate for activity. In contrast, HPP epoxidase is alpha-ketoglutarate independent. Thus, the cyclization of HPP to fosfomycin clearly represents an intriguing conversion beyond the scope entailed by common biological epoxidation and C-O bond formation.
磷霉素是一种临床常用抗生素，其生物合成的最后一步是将（S）-2-羟丙基膦酸（HPP）转化为磷霉素。由于在早期的饲喂实验中已表明磷霉素中的环氧基源自HPP的羟基，因此这种环氧乙烷形成反应实际上是一个脱氢过程。为了研究这一独特的C-O键形成步骤，我们对所需的HPP环氧化酶进行了过表达和纯化。本文报道的结果提供了初步的生化证据，表明HPP环氧化酶是一种铁依赖性酶，其活性需要NAD（P）H和黄素或黄素蛋白还原酶。氧化态铁重构的磷霉素环氧化酶的2K EPR光谱显示，在至少两种环境中存在典型的S =（5）/（2）Fe（III）中心共振。添加HPP会导致重新分布，并出现至少两种其他物种，表明铁环境受到扰动。将该样品暴露于NO中没有引起变化，表明铁几乎全部处于Fe（III）状态。然而，向未暴露于O（2）的Fe（II）重构酶中添加NO会产生典型的S =（3）/（2）Fe（II）-NO复合物的强烈EPR光谱。该复合物也是异质的，但添加底物会将其转化为具有新EPR光谱的单一、均质的S =（3）/（2）物种，表明底物与铁结合或在铁附近结合，从而使中心有序排列。NO与亚铁中心结合这一事实表明，O（2）也可以作为催化循环的一部分在该位点结合。使用纯化的环氧化酶和（18）O同位素标记的HPP，证明了HPP的羟基氧在磷霉素中的保留。虽然仲醇脱氢形成醚环在文献中有先例，但这些催化作用需要α-酮戊二酸才能发挥活性。相比之下，HPP环氧化酶不依赖α-酮戊二酸。因此，HPP环化生成磷霉素显然代表了一种超出常见生物环氧化和C-O键形成范围的有趣转化。
