NDM-1金属β-内酰胺酶实验室突变体的动力学研究及35位异亮氨酸的重要性
Kinetic Study of Laboratory Mutants of NDM-1 Metallo-β-Lactamase and the Importance of an Isoleucine at Position 35.
作者信息
Marcoccia Francesca, Bottoni Carlo, Sabatini Alessia, Colapietro Martina, Mercuri Paola Sandra, Galleni Moreno, Kerff Frédéric, Matagne André, Celenza Giuseppe, Amicosante Gianfranco, Perilli Mariagrazia
机构信息
Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università degli Studi dell'Aquila, L'Aquila, Italy.
Laboratoire de Macromolécules Biologiques, Centre d'Ingénierie des Protéines, Université de Liège, Liège, Belgium.
出版信息
Antimicrob Agents Chemother. 2016 Mar 25;60(4):2366-72. doi: 10.1128/AAC.00531-15. Print 2016 Apr.
Abstract
Two laboratory mutants of NDM-1 were generated by replacing the isoleucine at position 35 with threonine and serine residues: the NDM-1(I35T)and NDM-1(I35S)enzymes. These mutants were well characterized, and their kinetic parameters were compared with those of the NDM-1 wild type. Thekcat,Km, andkcat/Kmvalues calculated for the two mutants were slightly different from those of the wild-type enzyme. Interestingly, thekcat/Kmof NDM-1(I35S)for loracarbef was about 14-fold higher than that of NDM-1. Far-UV circular dichroism (CD) spectra of NDM-1 and NDM-1(I35T)and NDM-1(I35S)enzymes suggest local structural rearrangements in the secondary structure with a marked reduction of α-helix content in the mutants.
摘要
通过将35位的异亮氨酸替换为苏氨酸和丝氨酸残基，生成了两种NDM-1实验室突变体：NDM-1（I35T）和NDM-1（I35S）酶。对这些突变体进行了充分表征，并将其动力学参数与NDM-1野生型的参数进行了比较。计算得出的两种突变体的kcat、Km和kcat/Km值与野生型酶的这些值略有不同。有趣的是，NDM-1（I35S）对氯碳头孢的kcat/Km比NDM-1高约14倍。NDM-1、NDM-1（I35T）和NDM-1（I35S）酶的远紫外圆二色（CD）光谱表明二级结构发生了局部结构重排，突变体中的α-螺旋含量显著降低。
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