Protein purification and function assignment of the epoxidase catalyzing the formation of fosfomycin
Protein purification and function assignment of the epoxidase catalyzing the formation of fosfomycin
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DOI：
10.1021/ja004153y
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发表时间：
2001-05-16
影响因子：
15
通讯作者：
Liu, HW
中科院分区：
文献类型：
--
作者：
Liu, PH;Murakami, K;Liu, HW
(1R，2S）-环氧丙基膦酸（1），也称为磷霉素，是临床上有用的抗生素。1它的生物学作用靶点被确定为UDP-GlcNAc-O-烯醇式丙酮酸转移酶，2催化磷酸烯醇式丙酮酸（PEP）与UDP-GlcNAc的结合，3是细菌细胞壁肽聚糖层组装的关键步骤。早期的研究表明磷霉素的生物合成始于PEP（2）异构化为膦酰丙酮酸（3）（方案1）。4饲养实验还确定磷霉素的直接前体是（S）-2-羟丙基膦酸（HPP，4）。5基于这些发现，提出了磷霉素生物合成的最少四个酶促步骤（方案1）。6最近，濑户和同事们从韦氏链霉菌7中克隆了整个磷霉素生物合成基因簇，并从假单胞菌PB-5123中克隆了部分基因簇。8.在大肠杆菌中表达P. dingae的orf 3并进行初步活性测定，结果初步确定orf 3编码HPP环氧酶。[8]在一次共同的努力中，我们已经用S表示了orf 3的等价物。wedmorensis（fom 4）中表达，并纯化了其编码的蛋白（Fom 4）。此外，我们还开发了一种有效的Fom 4测定法，现在允许首次明确指定Fom 4为所需的HPP环氧酶。本文报道了这种酶的初步特征及其催化模式的影响。通过聚合酶链反应（PCR）扩增Fom 4基因，并克隆到pQE 30表达载体中。随后的质粒pQE 5110用于转化E. coli M15宿主细胞。重组菌在LB培养基中的生长导致Fom 4的过量产生。该N-末端His-标记的酶被纯化，并且在300 nm以上没有表现出明显的吸收。这种纯化蛋白的电感耦合等离子体（ICP）分析也未能检测到任何氧化还原活性金属离子的显着数量。为了确定Fom 4是否是期望的环氧酶，合成（S）-HPP（4），9，10并测试其作为Fom 4底物的能力。令人失望的是，在该孵育中，通过NMR不能检测到磷霉素。然后使用生物测定8来评估Fom 4作为环氧酶发挥功能是否需要各种金属离子和常见的生物还原剂。11在该实验中，将浸有测定混合物的纸片与大肠杆菌菌苔直接接触。coli K12 HW 8235在营养琼脂（LB）上生长。当测定混合物中存在磷霉素时，孵育几小时后可见抑制区。利用这种灵敏的生物自显影方法，发现Fe（NH_4）_2（SO_4）_2和NAD（P）H都是Fom_4将4转化为磷霉素所必需的。这些发现提供了初步证据，表明Fom 4是所需的环氧酶，其催化作用是铁依赖性的。然而，即使在孵育中包括所有上述组分时，也没有通过NMR可辨别的磷霉素产生。由于该环氧酶是铁依赖性的，为了避免可能与融合的His 6-标签相关的任何并发症，将fom 4基因克隆到pET 24 b载体中以表达其野生型形式的环氧酶。13纯化的蛋白质作为具有89 kDa的表观Mr的单峰从FPLC S200柱洗脱，并且基于21210 Da的单体Mr计算为
(1R, 2S)-Epoxypropylphosphonic acid (1), also known as fosfomycin, is a clinically useful antibiotic. 1 Its biological target has been identified as UDP-GlcNAc-O-enolpyruvoyl transferase, 2 which catalyzes the attachment of phosphoenolpyruvate (PEP) to UDP-GlcNAc, 3 a key step in the assembly of the peptidoglycan layer in bacterial cell wall. Early studies had shown that the biosynthesis of fosfomycin begins with isomerization of PEP (2) to phosphonopyruvate (3)(Scheme 1). 4 Feeding experiments also established that the immediate precursor of fosfomycin is (S)-2-hydroxypropylphosphonic acid (HPP, 4). 5 On the basis of these findings, a minimum of four enzymatic steps had been proposed for the biogenesis of fosfomycin (Scheme 1). 6 Recently, Seto and co-workers had cloned the entire fosfomycin biosynthetic gene cluster from Streptomyces wedmorensis, 7 and part of the cluster from Pseudomonas syringae PB-5123. 8 Expression of orf3 of P. syringae in Escherichia coli and a preliminary activity assay led to the tentative assignment of orf3 as encoding for the HPP epoxidase. 8 In a joint effort, we have expressed the orf3 equivalent in S. wedmorensis (fom4) and purified the encoded protein (Fom4). In addition, we have also developed an efficient assay for Fom4, which now allows the first unambiguous assignment of Fom4 as the desired HPP epoxidase. Reported herein are the initial characterization of this enzyme and the implications for its mode of catalysis.The gene for Fom4 was amplified by polymerase chain reaction (PCR) and cloned into a pQE30 expression vector. The ensuing plasmid, pQE5110, was used to transform E. coli M15 host cells. Growth of the recombinant strain in LB medium led to overproduction of Fom4. This N-terminal His-tagged enzyme was purified and exhibited no apparent absorption above 300 nm. Inductive coupled plasma (ICP) analysis of this purified protein also failed to detect any redox-active metal ions in significant quantities. To determine whether Fom4 is the desired epoxidase,(S)-HPP (4) was synthesized, 9, 10 and tested for its competence as the substrate for Fom4. To our disappointment, no fosfomycin could be detected by NMR in this incubation. A bioassay8 was then used to evaluate whether various metal ions and common biological reducing agents are required for Fom4 to function as an epoxidase. 11 In this experiment, a paper disk soaked with the assay mixture was placed in direct contact with a lawn of E. coli K12 HW8235 grown on nutrient (LB) agar. When fosfomycin was present in the assay mixture, an inhibition zone was visible after a few hours of incubation. Using this sensitive bioautography method, it was found that both Fe (NH4) 2 (SO4) 2 and NAD (P) H are essential for Fom4 to convert 4 to fosfomycin. These findings provided initial evidence revealing that Fom4 is the desired epoxidase, and its catalysis is iron-dependent. However, no fosfomycin production was discernible by NMR even when all of the above components were included in the incubation. 12 Since this epoxidase is iron-dependent, to avert any complication that may be associated with the fused His6-tag, the fom4 gene was cloned into a pET24b vector to express the epoxidase in its wild-type form. 13 The purified protein was eluted as a single peak with an apparent Mr of 89 kDa from an FPLC S200 column, and was calculated based on a monomeric Mr of 21210 Da to be