X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria
X-ray absorption spectroscopy of the zinc-binding sites in the class B2 metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria
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DOI：
10.1021/bi061547e
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发表时间：
2006-11-14
期刊：
影响因子：
2.9
通讯作者：
Tierney, David L.
中科院分区：
文献类型：
--
作者：
Costello, Alison L.;Sharma, Narayan P.;Tierney, David L.
用X射线吸收光谱研究了Veronii气单胞菌IMIS的金属结合部位。温和剂有催化活性(1-锌)、产物抑制(1-锌加亚胺培南)和非活性(2-锌)形式。第一个等量的锌(II)被发现与共识的锌-2结合。1-锌IMIS与亚胺培南反应生成产物结合物种，通过羧酸基与锌配位。通过野生型IMIS与1当量和2当量结合锌的比较，检测IMIS的抑制结合部位。2-锌IMIS扩展X射线吸收精细结构数据支持一个远离活性中心的结合位置，并且包含一个硫供体和一个组氨酸配体。根据IMIS的氨基酸序列和CPHA的晶体结构[Garau et al.(2005)J.Mol.比奥尔。345,785-795]，我们认为抑制结合部位是由位于B2不同的α3螺旋上的M146和H118形成的，H118是一种典型的锌-1配体，有助于激活亲核水。M146突变为异亮氨酸可消除金属抑制。这是第一次用天然金属锌-1表征综管信息系统，并首次确定了抑制金属的位置。
X- ray absorption spectroscopy was used to investigate the metal- binding sites of ImiS from Aeromonas Veronii bV. sobria in catalytically active ( 1-Zn), product- inhibited ( 1-Zn plus imipenem), and inactive ( 2-Zn) forms. The first equivalent of zinc(II) was found to bind to the consensus Zn-2 site. The reaction of 1- Zn ImiS with imipenem leads to a product- bound species, coordinated to Zn via a carboxylate group. The inhibitory binding site of ImiS was examined by a comparison of wild- type ImiS with 1 and 2 equiv of bound zinc. 2- Zn ImiS extended X- ray absorption fine structure data support a binding site that is distant from the active site and contains both one sulfur donor and one histidine ligand. On the basis of the amino acid sequence of ImiS and the crystal structure of CphA [ Garau et al. ( 2005) J. Mol. Biol. 345, 785- 795], we propose that the inhibitory binding site is formed by M146, found on the B2distinct alpha 3 helix, and H118, a canonical Zn-1 ligand, proposed to help activate the nucleophilic water. The mutation of M146 to isoleucine abolishes metal inhibition. This is the first characterization of ImiS with the native metal Zn-1 and establishes, for the first time, the location of the inhibitory metal site.