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首页> 外文期刊>Journal of Molecular Biology >Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer--dimer equilibrium.
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Characterization and manipulation of the Pseudomonas aeruginosa dimethylarginine dimethylaminohydrolase monomer--dimer equilibrium.

机译：铜绿假单胞菌二甲基精氨酸二甲基氨基水解酶单体-二聚体平衡的表征和操作。

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In mammals, the enzyme dimethylarginine dimethylaminohydrolase (DDAH) is implicated in the regulation of the cellular levels of asymmetric methylarginines, small molecule metabolites that themselves represent a family of endogenous inhibitors of nitric oxide synthase (NOS). The involvement of DDAH function in the regulation of NOS makes this enzyme a potentially attractive therapeutic target. DDAH from the bacterium Pseudomonas aeruginosa (PaDDAH) is so far the only structurally tractable homologue of mammalian DDAH isoforms. To complement the recent crystal structure of this protein, we show by hydrodynamic measurements that PaDDAH exists in dynamic equilibrium between monomer (ca 29 kDa) and symmetric homodimer (ca 58 kDa) states with a dimer dissociation constant, K(d) approximately 500nM. For the purposes of NMR-based approaches to the study of this enzyme's interactions with substrate and inhibitor ligands, it would be useful to obtain the protein in monomeric form. Through detailed analysis of the homodimer PaDDAH crystal structure we identified key residues involved in the protomer-protomer interface and targeted these for mutation. The hydrodynamic and self-associative properties of a series of PaDDAH interface mutants were analyzed by concentration-dependent analytical size-exclusion chromatography and sedimentation equilibrium analytical ultracentrifugation. The individual substitution of several of the interface residues shifts the equilibrium position towards the monomer, which allowed the design of a double mutant variant (Arg40-->Glu, Arg98-->His) that behaves exclusively as a stable monomer, yet retains greater than 95% catalytic activity compared to wild-type. Comparative two-dimensional (1)H, (15)N heteronuclear NMR spectra indicate that the double mutant remains a monomer even at approximately 1 mM concentration. Accordingly, the double mutant PaDDAH is an attractive template for further NMR-based investigations of the enzyme mechanism and characterization of ligand-binding and inhibitor-binding profiles. These results indicate that dimerization of PaDDAH is not critical for the maintenance of the biological function of the protein. These results are discussed in the context of known modes of self-association between structurally related, but functionally distinct, members of the beta/alpha-propeller fold class.
机译：在哺乳动物中，二甲基精氨酸二甲基氨基水解酶（DDAH）参与不对称甲基精氨酸（小分子代谢产物的细胞水平的调节），这些分子本身代表一氧化氮合酶（NOS）的内源性抑制剂家族。 DDAH功能参与NOS的调节使该酶成为潜在有吸引力的治疗靶标。迄今为止，来自铜绿假单胞菌（PaDDAH）的DDAH是哺乳动物DDAH亚型的唯一在结构上易于处理的同源物。为了补充该蛋白质的最新晶体结构，我们通过流体力学测量表明，PaDDAH存在于单体（ca 29 kDa）和对称同型二聚体（ca 58 kDa）状态之间的动态平衡中，其中二聚解离常数K（d）约为500nM。出于基于NMR的方法研究该酶与底物和抑制剂配体相互作用的目的，获得单体形式的蛋白质将很有用。通过对同型二聚体PaDDAH晶体结构的详细分析，我们确定了与启动子-启动子界面有关的关键残基，并将其靶向突变。通过浓度依赖性分析尺寸排阻色谱法和沉降平衡分析超速离心法分析了一系列PaDDAH界面突变体的流体动力学和自缔合特性。多个界面残基的单独取代将平衡位置移向单体，从而允许设计一个双重突变变体（Arg40-> Glu，Arg98-> His），该变体仅表现为稳定的单体，但保留了更大的稳定性。与野生型相比，催化活性超过95％。比较的二维（1）H，（15）N异核NMR光谱表明，即使在大约1 mM的浓度下，双突变体仍然是单体。因此，双突变体PaDDAH是用于酶机理以及配体结合和抑制剂结合谱表征的进一步基于NMR的研究的有吸引力的模板。这些结果表明，PaDDAH的二聚化对于维持蛋白质的生物学功能不是至关重要的。这些结果是在结构相关但功能上不同的beta / alpha-propeller折叠类成员之间的已知自相关模式下讨论的。

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