Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.
Structure-Function Studies of the Antibiotic Target l,l-Diaminopimelate Aminotransferase from Verrucomicrobium spinosum Reveal an Unusual Oligomeric Structure.
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来自刺疣微菌 (Verrucomicrobium spinosum) 的抗生素靶点 l,l-二氨基庚二酸转氨酶的结构-功能研究揭示了一种不寻常的寡聚结构。
DOI：
10.1021/acs.biochem.0c00185
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Hudson,AndréO
中科院分区：
文献类型：
--
作者：
Weatherhead,AnthonyW;Crowther,JenniferM;Horne,ChristopherR;Meng,Yanxiang;Coombes,David;Currie,MichaelJ;Watkin,SerenaAJ;Adams,LilyE;Parthasarathy,Anutthaman;Dobson,RenwickCJ;Hudson,AndréO
虽然人类缺乏形成间二氨基庚二酸和l-赖氨酸的生物合成途径，但它们对细菌生存至关重要，因此是抗生素的有吸引力的靶标。最近发现衣原体家族成员利用赖氨酸生物合成途径中一种罕见的氨基转移酶途径，从而提供了一种新的酶促药物靶点。本文研究了沙眼衣原体非致病性模式菌刺疣微菌（VsDapL）的二氨基庚二酸氨基转移酶的特性。spinosum dapL基因编码真正的二氨基庚二酸氨基转移酶，因为该基因拯救了一种营养缺陷型的大肠杆菌菌株。动力学研究表明，VsDapL遵循Michaelis-Menten机理，其对底物l-二氨基庚二酸的Km为4.0mM。kcat（0.46 s-1）和kcat/KM（115 s-1 M-1）略低于其它二氨基庚二酸转氨酶的值。此外，尽管其他研究的DapL直向同源物是二聚体，沉降速度实验表明VsDapL以单体-二聚体自缔合形式存在，其KD 2 - 1为7.4 μM。2.25 μ m分辨率的晶体结构呈现典型的杯形单体的二聚体，并且小角X射线散射实验证实了溶液中的二聚体。序列和结构比对表明，活性位点残基的重要活动是保守的inVsDapL，尽管较低的活动相比，其他DapL同源物。虽然二聚体界面掩埋了总表面积的18%，但对界面和活性位点有贡献的几个环，特别是L1、L2和L5环，是高度移动的，这可能解释了不稳定的二聚体和较低的催化活性。我们的动力学，生物物理和结构表征可用于通知抗生素的发展。
While humans lack the biosynthetic pathways formeso-diaminopimelate andl-lysine, they are essential for bacterial survival and are therefore attractive targets for antibiotics. It was recently discovered that members of theChlamydiafamily utilize a rare aminotransferase route of thel-lysine biosynthetic pathway, thus offering a new enzymatic drug target. Here we characterize diaminopimelate aminotransferase fromVerrucomicrobium spinosum(VsDapL), a nonpathogenic model bacterium forChlamydia trachomatis.Complementation experiments verify that theV. spinosum dapLgene encodes a bona fide diaminopimelate aminotransferase, because the gene rescues anEscherichia colistrain that is auxotrophic formeso-diaminopimelate. Kinetic studies show thatVsDapL follows a Michaelis–Menten mechanism, with aKMappof 4.0 mM toward its substratel,l-diaminopimelate. Thekcat(0.46 s–1) and thekcat/KM(115 s–1M–1) are somewhat lower than values for other diaminopimelate aminotransferases. Moreover, whereas other studied DapL orthologs are dimeric, sedimentation velocity experiments demonstrate thatVsDapL exists in a monomer–dimer self-association, with aKD2-1of 7.4 μM. The 2.25 Å resolution crystal structure presents the canonical dimer of chalice-shaped monomers, and small-angle X-ray scattering experiments confirm the dimer in solution. Sequence and structural alignments reveal that active site residues important for activity are conserved inVsDapL, despite the lower activity compared to those of other DapL homologues. Although the dimer interface buries 18% of the total surface area, several loops that contribute to the interface and active site, notably the L1, L2, and L5 loops, are highly mobile, perhaps explaining the unstable dimer and lower catalytic activity. Our kinetic, biophysical, and structural characterization can be used to inform the development of antibiotics.
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DOI：
10.1111/j.1432-1033.1972.tb01832.x
发表时间：
1972
期刊：
European journal of biochemistry
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影响因子：
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作者：
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DOI：
--
发表时间：
1986
期刊：
Laboratory investigation; a journal of technical methods and pathology
影响因子：
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DOI：
--
发表时间：
1979
期刊：
影响因子：
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作者：
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