Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis
Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis
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DOI：
10.1074/jbc.m113.450148
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发表时间：
2013-03-29
影响因子：
4.8
通讯作者：
Perugini, Matthew A.
中科院分区：
文献类型：
--
作者：
Hor, Lilian;Dobson, Renwick C. J.;Perugini, Matthew A.
二氨基庚二酸（DAP）差向异构酶参与meso-DAP和赖氨酸的生物合成，它们是合成肽聚糖、管家蛋白和细菌毒力因子的重要前体。因此，DAP差向异构酶是一个有前途的抗菌靶标。以往的研究报道DAP差向异构酶以单体酶的形式存在。然而，我们表明，使用分析超离心，X-射线晶体学，和酶动力学分析，DAP差向异构酶从大肠杆菌存在作为一个功能性的二聚体在溶液中和晶体状态。此外，E. coliDAP差向异构酶二聚体首次表明该酶以开放的活性构象存在。二聚化的重要性随后通过使用定点诱变产生单体突变体（Y268 A）来探测。我们的研究表明，Y268 A是无催化活性的，从而证明DAP差向异构酶的二聚化是催化所必需的。分子动力学模拟表明，DAP差向异构酶单体本质上比二聚体更灵活，这表明二聚化优化了蛋白质动力学以支持功能。我们的研究结果提供了深入了解新的抗菌药物的发展目标的二聚体抗生素目标DAP差向异构酶。
Diaminopimelate (DAP) epimerase is involved in the biosynthesis of meso-DAP and lysine, which are important precursors for the synthesis of peptidoglycan, housekeeping proteins, and virulence factors in bacteria. Accordingly, DAP epimerase is a promising antimicrobial target. Previous studies report that DAP epimerase exists as a monomeric enzyme. However, we show using analytical ultracentrifugation, X-ray crystallography, and enzyme kinetic analyses that DAP epimerase from Escherichia coli exists as a functional dimer in solution and the crystal state. Furthermore, the 2.0-angstrom X-ray crystal structure of the E. coli DAP epimerase dimer shows for the first time that the enzyme exists in an open, active conformation. The importance of dimerization was subsequently probed by using site-directed mutagenesis to generate a monomeric mutant (Y268A). Our studies show that Y268A is catalytically inactive, thus demonstrating that dimerization of DAP epimerase is essential for catalysis. Molecular dynamics simulations indicate that the DAP epimerase monomer is inherently more flexible than the dimer, suggesting that dimerization optimizes protein dynamics to support function. Our findings offer insight into the development of novel antimicrobial agents targeting the dimeric antibiotic target DAP epimerase.