Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-β-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution
Difference in the Inhibitory Effect of Thiol Compounds and Demetallation Rates from the Zn(II) Active Site of Metallo-β-lactamases (IMP-1 and IMP-6) Associated with a Single Amino Acid Substitution
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与单一氨基酸取代相关的硫醇化合物的抑制效果和金属-β-内酰胺酶（IMP-1 和 IMP-6）Zn(II) 活性位点的脱金属率的差异
DOI：
10.1021/acsinfecdis.2c00395
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发表时间：
2022
影响因子：
5.3
通讯作者：
Kurosaki Hir
中科院分区：
文献类型：
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作者：
Yamaguchi Yoshihiro;Kato Koichi;Ichimaru Yoshimi;Uenosono Yuya;Tawara Sakiko;Ito Rio;Matsuse Natsuki;Wachino Jun-ichi;Toma-Fukai Sachiko;Jin Wanchun;Arakawa Yoshichika;Otsuka Masami;Fujita Mikako;Fukuishi Nobuyuki;Sugiura Kirara;Imai Masanori;Kurosaki Hir
产金属β-内酰胺酶（MBL）的革兰阴性菌已成为公共卫生的一大威胁。包括IMP、Vim和NDM类型的MBL是Zn（II）酶，其水解存在于广泛的抗生素中的β-内酰胺环，例如N-苄青霉素、美罗培南和亚胺培南。在IMP中，IMP-1和IMP-6的不同之处在于位置262处的单个氨基酸取代，其中IMP-1中的丝氨酸被IMP-6中的甘氨酸取代，从而赋予底物特异性的变化。为了研究这种突变如何影响酶的功能，我们研究了巯基化合物对内酰胺酶的抑制作用。3-巯基丙酸乙酯是IMP-1的竞争性抑制剂，但对IMP-6是非竞争性抑制剂。比较先前报道的IMP-1（PDB代码：5EV 6）和IMP-6（PDB代码：6LVJ）的晶体结构，揭示了IMP-1中Ser 262和Cys 221侧链之间的氢键，但IMP-6中不存在氢键，这影响了其活性位点中的Zn 2配位球。研究了在乙二胺四乙酸（EDTA）存在下IMP-1和IMP-6的脱金属反应，发现脱金属反应有快、慢两个阶段，其一级反应速率常数分别为：IMP-1的kfast= 1.76h-1，kslow= 0.108h-1; IMP-6的kfast = 14.0h-1，kslow = 1.66h-1。IMP-1和IMP-6之间Zn 2配位球柔性的差异可能影响脱金属速率、对β-内酰胺抗生素的催化效率以及硫醇化合物的抑制效果。
Gram-negative bacteria producing metallo-β-lactamases (MBLs) have become a considerable threat to public health. MBLs including the IMP, VIM, and NDM types are Zn(II) enzymes that hydrolyze the β-lactam ring present in a broad range of antibiotics, such asN-benzylpenicillin, meropenem, and imipenem. Among IMPs, IMP-1 and IMP-6 differ in a single amino acid substitution at position 262, where serine in IMP-1 is replaced by glycine in IMP-6, conferring a change in substrate specificity. To investigate how this mutation influences enzyme function, we examined lactamase inhibition by thiol compounds. Ethyl 3-mercaptopropionate acted as a competitive inhibitor of IMP-1, but a noncompetitive inhibitor of IMP-6. A comparison of the crystal structures previously reported for IMP-1 (PDB code: 5EV6) and IMP-6 (PDB code: 6LVJ) revealed a hydrogen bond between the side chain of Ser262 and Cys221 in IMP-1 but the absence of hydrogen bond in IMP-6, which affects the Zn2 coordination sphere in its active site. We investigated the demetallation rates of IMP-1 and IMP-6 in the presence of chelating agent ethylenediaminetetraacetic acid (EDTA) and found that the demetallation reactions had fast and slow phases with a first-order rate constant (kfast= 1.76 h–1,kslow= 0.108 h–1for IMP-1, andkfast= 14.0 h–1andkslow= 1.66 h–1for IMP-6). The difference in the flexibility of the Zn2 coordination sphere between IMP-1 and IMP-6 may influence the demetallation rate, the catalytic efficiency against β-lactam antibiotics, and the inhibitory effect of thiol compounds.