Ely Fernanda, Nunes José E S, Schroeder Evelyn K, Frazzon Jeverson, Palma Mário S, Santos Diógenes S, Basso Luiz A
Centro de Pesquisas em Biologia Molecular e Funcional, Pontifícia Universidade Católica do Rio Grande do Sul, RS 90619-900, Porto Alegre, Brazil.
BMC Biochem. 2008 Apr 29;9:13. doi: 10.1186/1471-2091-9-13.
The emergence of multi- and extensively-drug resistant Mycobacterium tuberculosis strains has created an urgent need for new agents to treat tuberculosis (TB). The enzymes of shikimate pathway are attractive targets to the development of antitubercular agents because it is essential for M. tuberculosis and is absent from humans. Chorismate synthase (CS) is the seventh enzyme of this route and catalyzes the NADH- and FMN-dependent synthesis of chorismate, a precursor of aromatic amino acids, naphthoquinones, menaquinones, and mycobactins. Although the M. tuberculosis Rv2540c (aroF) sequence has been annotated to encode a chorismate synthase, there has been no report on its correct assignment and functional characterization of its protein product.
In the present work, we describe DNA amplification of aroF-encoded CS from M. tuberculosis (MtCS), molecular cloning, protein expression, and purification to homogeneity. N-terminal amino acid sequencing, mass spectrometry and gel filtration chromatography were employed to determine identity, subunit molecular weight and oligomeric state in solution of homogeneous recombinant MtCS. The bifunctionality of MtCS was determined by measurements of both chorismate synthase and NADH:FMN oxidoreductase activities. The flavin reductase activity was characterized, showing the existence of a complex between FMNox and MtCS. FMNox and NADH equilibrium binding was measured. Primary deuterium, solvent and multiple kinetic isotope effects are described and suggest distinct steps for hydride and proton transfers, with the former being more rate-limiting.
This is the first report showing that a bacterial CS is bifunctional. Primary deuterium kinetic isotope effects show that C4-proS hydrogen is being transferred during the reduction of FMNox by NADH and that hydride transfer contributes significantly to the rate-limiting step of FMN reduction reaction. Solvent kinetic isotope effects and proton inventory results indicate that proton transfer from solvent partially limits the rate of FMN reduction and that a single proton transfer gives rise to the observed solvent isotope effect. Multiple isotope effects suggest a stepwise mechanism for the reduction of FMNox. The results on enzyme kinetics described here provide evidence for the mode of action of MtCS and should thus pave the way for the rational design of antitubercular agents.
多重耐药和广泛耐药结核分枝杆菌菌株的出现，迫切需要新的药物来治疗结核病（TB）。莽草酸途径的酶是开发抗结核药物的有吸引力的靶点，因为它对结核分枝杆菌至关重要，而在人类中不存在。分支酸合酶（CS）是该途径的第七种酶，催化依赖NADH和FMN的分支酸合成，分支酸是芳香族氨基酸、萘醌、甲萘醌和分枝杆菌素的前体。尽管结核分枝杆菌Rv2540c（aroF）序列已被注释为编码分支酸合酶，但尚未有关于其正确归属及其蛋白质产物功能表征的报道。
在本研究中，我们描述了从结核分枝杆菌（MtCS）中扩增aroF编码的CS的DNA、分子克隆、蛋白质表达以及纯化至均一性。采用N端氨基酸测序、质谱和凝胶过滤色谱法来确定均一重组MtCS在溶液中的身份、亚基分子量和寡聚状态。通过测量分支酸合酶和NADH：FMN氧化还原酶活性来确定MtCS的双功能特性。对黄素还原酶活性进行了表征，表明FMNox与MtCS之间存在复合物。测量了FMNox和NADH的平衡结合。描述了初级氘、溶剂和多重动力学同位素效应，表明氢化物和质子转移存在不同步骤，前者是更限速的步骤。
这是首次报道表明细菌CS具有双功能。初级氘动力学同位素效应表明，在NADH还原FMNox的过程中，C4-proS氢正在转移，并且氢化物转移对FMN还原反应的限速步骤有显著贡献。溶剂动力学同位素效应和质子总量结果表明，来自溶剂的质子转移部分限制了FMN还原速率，并且单个质子转移产生了观察到的溶剂同位素效应。多重同位素效应表明FMNox还原存在逐步机制。此处描述的酶动力学结果为MtCS的作用模式提供了证据，因此应为抗结核药物的合理设计铺平道路。
