Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159
Heterologous expression, purification and biochemical characterization of a glutamate racemase (MurI) from Streptococcus mutans UA159
复制标题
变形链球菌 UA159 谷氨酸消旋酶 (MurI) 的异源表达、纯化和生化表征
DOI：
10.7717/peerj.8300
复制
发表时间：
2019-12-20
期刊：
影响因子：
2.7
通讯作者：
Zhang，Jiangying
中科院分区：
文献类型：
--
作者：
Wang，Xiangzhu;Chen，Chanchan;Zhang，Jiangying
背景谷氨酸消旋酶（MurI）是一种不依赖于辅因子的酶，在细菌肽聚糖生物合成途径中起重要作用，因此被认为是开发抗菌药物的一个有吸引力的靶点。虽然在我们以前的研究中，murI基因的必要性被证明在变形链球菌，人类龋齿的主要病原体，研究S。mutans MurI尚未提供确切结果。本研究的目的是从S.变形杆菌UA 159基因组。方法采用生物信息学方法进行结构预测和多序列比对。重组His 6标记的S.在表达载体pColdII中过表达变形菌MurI，并使用Ni 2+亲和层析法进一步纯化。通过SDS-PAGE、Western blotting、native PAGE和SEC-HPLC分析蛋白质的溶解性、纯度和聚集状态。通过圆二色性（CD）测定来评估动力学参数。基于Michaelis-Menten方程的曲线拟合计算动力学常数。温度和pH对酶活性的影响通过一系列偶联酶反应混合物来确定。结果获得了S.用PCR方法扩增了变形杆菌UA 159基因，克隆并在大肠杆菌BL 21（DE 3）中表达。264个氨基酸的蛋白质，作为二聚体和单体酶的混合物，被纯化至电泳均一性。在CD测定中，S.变异株MurI显示出独特的动力学参数（Km，d-Glu→l-Glu = 0.3631 ± 0.3205 mM，Vmax，d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1，kcat，d-Glu→l-Glu = 0.0306 ± 0.0065 s−1，kcat/Km，d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1，以d-谷氨酸为底物; Km，l-Glu→d-Glu = 0.8077 ± 0.5081 mM，Vmax，l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1，kcat，l-Glu→d-Glu = 0.0378 ± 0.0056 s−1，kcat/Km，l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1，以l-谷氨酸为底物）。S.变形杆菌MurI的最适测定温度为37.5 °C，最适pH为8.0。结论本研究结果为进一步了解S.为谷氨酸消旋酶在抗龋药物设计中的应用提供了一个可行的指导。
Background Glutamate racemase (MurI) is a cofactor-independent enzyme that is essential to the bacterial peptidoglycan biosynthesis pathway and has therefore been considered an attractive target for the development of antimicrobial drugs. While in our previous study the essentiality of the murI gene was shown in Streptococcus mutans, the primary aetiologic agent of human dental caries, studies on S. mutans MurI have not yet provided definitive results. This study aimed to produce and characterize the biochemical properties of the MurI from the S. mutans UA159 genome. Methods Structure characterization prediction and multiple sequence alignment were performed by bioinformatic analysis. Recombinant His6-tagged S. mutans MurI was overexpressed in the expression vector pColdII and further purified using a Ni2+ affinity chromatography method. Protein solubility, purity and aggregation state were analyzed by SDS–PAGE, Western blotting, native PAGE and SEC-HPLC. Kinetic parameters were assessed by a circular dichroism (CD) assay. Kinetic constants were calculated based on the curve fit for the Michaelis–Menten equation. The effects of temperature and pH on enzymatic activity were determined by a series of coupled enzyme reaction mixtures. Results The glutamate racemase gene from S. mutans UA159 was amplified by PCR, cloned and expressed in Escherichia coli BL21 (DE3). The 264-amino-acid protein, as a mixture of dimeric and monomeric enzymes, was purified to electrophoretic homogeneity. In the CD assay, S. mutans MurI displayed unique kinetic parameters (Km, d-Glu→l-Glu = 0.3631 ± 0.3205 mM, Vmax, d-Glu→l-Glu = 0.1963 ± 0.0361 mM min−1, kcat, d-Glu→l-Glu = 0.0306 ± 0.0065 s−1, kcat/Km, d-Glu→l-Glu = 0.0844 ± 0.0128 s−1 mM−1, with d-glutamate as substrate; Km, l-Glu→d-Glu = 0.8077 ± 0.5081 mM, Vmax, l-Glu→d-Glu = 0.2421 ± 0.0418 mM min−1, kcat, l-Glu→d-Glu = 0.0378 ± 0.0056 s−1, kcat/Km, l-Glu→d-Glu = 0.0468 ± 0.0176 s−1 mM−1, with l-glutamate as substrate). S. mutans MurI possessed an assay temperature optimum of 37.5 °C and its optimum pH was 8.0. Conclusion The findings of this study provide insight into the structure and biochemical traits of the glutamate racemase in S. mutans and supply a conceivable guideline for employing glutamate racemase in anti-caries drug design.