The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis
The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis
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DOI：
10.1016/j.jmb.2005.07.045
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发表时间：
2005-09-16
影响因子：
5.6
通讯作者：
Rubio, V
中科院分区：
文献类型：
--
作者：
Marco-Marín, C;Gil-Ortiz, F;Rubio, V
UMP 激酶 (UMPK) 是负责微生物 UMP 磷酸化的酶，在嘧啶核苷酸生物合成中发挥着关键作用，通过反馈控制和氨基甲酰磷酸合成酶（嘧啶生物合成途径的第一种酶）的基因抑制来调节这一过程。我们展示了强烈火球菌 UMPK 的晶体结构，无论是游离的还是与 AMPPNP 或 AMPPNP 和 UMP 复合的，分辨率分别为 2.4 埃、3 埃和 2.55 埃，提供了具有催化能力的双底物复合物的真实快照。该结构证明 UMPK 与其他核苷单磷酸激酶（包括在动物中发现的 UMP/CMP 激酶）不同，因此 UMPK 可能是潜在的抗菌靶点。这种酶具有以中空核为中心的同六聚体结构，并被组织为二聚体的三聚体。 UMPK多肽表现出已在氨基甲酸酯激酶和乙酰谷氨酸激酶中报道的氨基酸激酶家族(AAKF)折叠。与乙酰谷氨酸激酶的比较表明，底物在每个亚基内的等效、充分适应的位点结合。 UMPK 结构包含两个结合的 Mg 离子，其中一个有助于稳定过渡态，因此与乙酰谷氨酸激酶中发现的一个赖氨酸残基具有相同的催化作用，而 P. Furiosus UMPK 中缺少该赖氨酸残基。相对于氨基甲酸酯激酶和乙酰谷氨酸激酶，UMPK 呈现出完全不同的二聚体结构，缺乏被认为是 AAKF 酶特征的特征性 16 链 β-折叠主链。其六聚体结构也是一个新特征，是由相邻二聚体的 A 和 B 亚基之间的赤道接触以及 A 或 B 亚基之间的极性接触组合产生的，并且可能是 UMPK 调节功能所必需的，例如基因调节，本文提出通过与 DNA 结合蛋白 PepA 的六聚体-六聚体相互作用介导。 (c) 2005 Elsevier Ltd. 保留所有权利。
UMP kinase (UMPK), the enzyme responsible for microbial UMP phosphorylation, plays a key role in pyrimidine nucleotide biosynthesis, regulating this process via feed-back control and via gene repression of carbamoyl phosphate synthetase (the first enzyme of the pyrimidine biosynthesis pathway). We present crystal structures of Pyrococcus furiosus UMPK, free or complexed with AMPPNP or AMPPNP and UMP, at 2.4 angstrom, 3 angstrom and 2.55 angstrom resolution, respectively, providing a true snapshot of the catalytically competent bisubstrate complex. The structure proves that UMPK does not resemble other nucleoside monophosphate kinases, including the UMP/CMP kinase found in animals, and thus UMPK may be a potential antimicrobial target. This enzyme has a homohexameric architecture centred around a hollow nucleus, and is organized as a trimer of dimers. The UMPK polypeptide exhibits the amino acid kinase family (AAKF) fold that has been reported in carbamate kinase and acetylglutamate kinase. Comparison with acetylglutamate kinase reveals that the substrates bind within each subunit at equivalent, adequately adapted sites. The UMPK structure contains two bound Mg ions, of which one helps stabilize the transition state, thus having the same catalytic role as one lysine residue found in acetylglutamate kinase, which is missing from P. furiosus UMPK. Relative to carbamate kinase and acetylglutamate kinase, UMPK presents a radically different dimer architecture, lacking the characteristic 16-stranded beta-sheet backbone that was considered a signature of AAKF enzymes. Its hexameric architecture, also a novel trait, results from equatorial contacts between the A and B subunits of adjacent dimers combined with polar contacts between A or B subunits and may be required for the UMPK regulatory functions, such as gene regulation, proposed here to be mediated by hexamer-hexamer interactions with the DNA-binding protein PepA. (c) 2005 Elsevier Ltd. All rights reserved.