Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1
Mechanistic and Spectroscopic Studies of Metallo-β-lactamase NDM-1
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DOI：
10.1021/bi300056y
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发表时间：
2012-05-08
期刊：
影响因子：
2.9
通讯作者：
Crowder, Michael W.
中科院分区：
文献类型：
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作者：
Yang, Hao;Aitha, Mahesh;Crowder, Michael W.
为了对金属-β-内酰胺酶NDM-1进行生物化学表征，我们克隆、过表达、纯化并表征了几种具有不同N-末端（全长、Delta 6、Delta 21和Delta 36）的麦芽糖结合蛋白（MBP）-NDM-1融合蛋白。所有MBP-NDM-1融合蛋白都是可溶的;然而，只有一种MBP-NDM-1 Delta 36表现出高活性并结合2当量的Zn（II）。凝血酶切割该融合蛋白产生截短的NDM-1 Delta 36变体，当使用头孢硝肟作为底物时，其表现出16 s（-1）的k（cat）和1.1 μ M的K-m，结合2当量的Zn（11），并且在溶液中是单体。NDM-1 Delta 36变体的扩展X射线吸收精细结构研究表明，该变体中Zn（II）的平均金属结合位点：由四个N/O供体（其中两个是组氨酸）和每个锌0.5个硫供体组成，Zn-Zn距离为3.38埃。该金属结合位点与属于B1亚类的其他金属β-内酰胺酶的金属结合位点非常相似。使用头孢硝肟和chromacef以及NDM-1 Delta 36变体进行的稳态前动力学研究表明，该酶利用与金属β-内酰胺酶L1和CcrA类似的动力学机制，其中活性氮阴离子被稳定，其质子化是限速的。虽然它们在氨基酸序列方面非常不同，但这些研究表明NDM-1在结构和机制上与金属β-内酰胺酶CcrA非常相似。
In an effort to biochemically characterize metallo-beta-lactamase NDM-1, we cloned, overexpressed, purified, and characterized several maltose binding protein (MBP)-NDM-1 fusion proteins with different N-termini (full-length, Delta 6, Delta 21, and Delta 36). All MBP-NDM-1 fusion proteins were soluble; however, only one, MBP NDM-1 Delta 36, exhibited high activity and bound 2 equiv of Zn(II). Thrombin cleavage of this fusion protein resulted in the truncated NDM-1 Delta 36 variant, which exhibited a k(cat) of 16 s(-1) and a K-m of 1.1 mu M when using nitrocefin as a substrate, bound 2 equiv of Zn(11), and was monomeric in solution. Extended X-ray absorption fine structure studies of the NDM-1 Delta 36 variant indicate the average metal binding site for Zn(II) in this variant: consists of four N/O donors (two of which are histidines) and 0.5 sulfur donor per zinc, with a Zn-Zn distance of 3.38 angstrom. This metal binding site is very similar to those of other metallo-beta-lactamases that belong to the B1 subclass. Pre-steady-state kinetic studies using nitrocefin and chromacef and the NDM-1 Delta 36 variant indicate that the enzyme utilizes a kinetic mechanism similar to that used by metallo-beta-lactamases L1 and CcrA, in which a reactive nitrogen anion is stabilized and its protonation is rate-limiting. While they are very different in terms of amino acid sequence, these studies demonstrate that NDM-1 is structurally and mechanistically very similar to metallo-beta-lactamase CcrA.