Family I soluble inorganic pyrophosphatases (PPases; EC 3.6.1.1) are enzymes essential for all organisms. They hydrolyze inorganic pyrophosphate, thus providing the driving force for numerous biosynthetic reactions. Soluble PPases retain enzymatic activity only in multimeric forms. PPases from various organisms are extensively studied by X-ray crystallography but until now there was no information on their structure and dynamics in solution. Hexameric 110 kDa (6 × 18.3 kDa) PPase from Mycobacterium tuberculosis (Mt-PPase) is a promising target for the rational design of potential anti-tuberculosis agents. In order to use NMR techniques in functional studies of Mt-PPase and rational design of the inhibitors for this enzyme, it is necessary to have information on the backbone ¹H, ¹³C and ¹⁵N resonance assignments. Samples of Mt-PPase enriched with 99% of ¹³C and ¹⁵N isotopes, and 95% of ²H were obtained using recombinant protein expression in an isotopically-labeled medium and effective heat-shock protocol for the deuterium-to-hydrogen exchange of the amide groups. Backbone resonance assignment was achieved for more than 95% of the residues. It was found that the secondary structure of Mt-PPase in solution corresponds well to the crystal structure of this protein. Protein backbone dynamics were studied using ¹⁵N NMR relaxation experiments. Determined resonance assignments and dynamic properties provide the basis for the subsequent structure-based design of novel inhibitors of Mt-PPase—potential anti-tuberculosis drugs.

中文翻译：

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来自结核分枝杆菌的 110 kDa 六聚无机焦磷酸酶的骨架共振分配和动力学。

家族 I 可溶性无机焦磷酸酶 (PPases; EC 3.6.1.1) 是所有生物体必不可少的酶。它们水解无机焦磷酸盐，从而为许多生物合成反应提供驱动力。可溶性 PPase 仅以多聚体形式保留酶活性。来自各种生物的 PPase 通过 X 射线晶体学进行了广泛的研究，但直到现在还没有关于它们在溶液中的结构和动力学的信息。来自结核分枝杆菌的六聚体 110 kDa (6 × 18.3 kDa) PPase (Mt-PPase) 是合理设计潜在抗结核药物的有希望的目标。为了在 Mt-PPase 的功能研究中使用 NMR 技术并合理设计该酶的抑制剂，有必要获得有关主链¹ H 的信息，¹³ C 和¹⁵ N 共振分配。Mt-PPase 的样品富含 99% 的¹³ C 和¹⁵ N 同位素，以及 95% 的² H 是通过在同位素标记的培养基中表达重组蛋白和有效的热激方案获得的，用于氘-氢交换酰胺基团。超过 95% 的残基实现了骨架共振分配。发现溶液中 Mt-PPase 的二级结构与该蛋白质的晶体结构非常吻合。使用¹⁵N NMR弛豫实验。确定的共振分配和动态特性为随后基于结构的新型 Mt-PPase 抑制剂（潜在的抗结核药物）设计提供了基础。