Yan Feng, Li Tingfeng, Lipscomb John D, Liu Aimin, Liu Hung-wen
Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX 78712, USA.
Arch Biochem Biophys. 2005 Oct 1;442(1):82-91. doi: 10.1016/j.abb.2005.07.024.
(S)-2-Hydroxylpropanylphosphonic acid epoxidase (HppE) is a novel type of mononuclear non-heme iron-dependent enzyme that catalyzes the O2 coupled, oxidative epoxide ring closure of HPP to form fosfomycin, which is a clinically useful antibiotic. Sequence alignment of the only two known HppE sequences led to the speculation that the conserved residues His138, Glu142, and His180 are the metal binding ligands of the Streptomyces wedmorensis enzyme. Substitution of these residues with alanine resulted in significant reduction of metal binding affinity, as indicated by EPR analysis of the enzyme-Fe(II)-substrate-nitrosyl complex and the spectral properties of the Cu(II)-reconstituted mutant proteins. The catalytic activities for both epoxidation and self-hydroxylation were also either eliminated or diminished in proportion to the iron content in these mutants. The complete loss of enzymatic activity for the E142A and H180A mutants in vivo and in vitro is consistent with the postulated roles of the altered residues in metal binding. The H138A mutant is also inactive in vivo, but in vitro it retains 27% of the active site iron and nearly 20% of the wild-type activity. Thus, it cannot be unequivocally stated whether H138 is an iron ligand or simply facilitates iron binding due to proximity. The results reported herein provide initial evidence implicating an unusual histidine/carboxylate iron ligation in HppE. By analogy with other well-characterized enzymes from the 2-His-1-carboxylate family, this type of iron core is consistent with a mechanism in which both oxygen and HPP bind to the iron as a first step in the in the conversion of HPP to fosfomycin.
（S）-2-羟基丙基膦酸环氧化酶（HppE）是一种新型的单核非血红素铁依赖性酶，它催化HPP与O2偶联的氧化环化反应以形成磷霉素，磷霉素是一种临床常用抗生素。对仅有的两个已知HppE序列进行序列比对后推测，保守残基His138、Glu142和His180是韦氏链霉菌酶的金属结合配体。用丙氨酸取代这些残基导致金属结合亲和力显著降低，这通过对酶-Fe（II）-底物-亚硝酰络合物的电子顺磁共振分析以及铜（II）重构突变蛋白的光谱特性得以表明。这些突变体中的环氧化和自羟基化催化活性也与铁含量成比例地消除或降低。E142A和H180A突变体在体内和体外酶活性的完全丧失与金属结合中改变的残基的假定作用一致。H138A突变体在体内也无活性，但在体外它保留了27%的活性位点铁和近20%的野生型活性。因此，不能明确说明His138是铁配体还是仅仅由于位置靠近而促进铁结合。本文报道的结果提供了初步证据，表明HppE中存在一种不寻常的组氨酸/羧酸盐铁配位。与2-组氨酸-1-羧酸盐家族的其他特征明确的酶类似，这种类型的铁核心与一种机制一致，即在HPP转化为磷霉素的第一步中，氧和HPP都与铁结合。
