Morin Sébastien, Clouthier Christopher M, Gobeil Sophie, Pelletier Joelle N, Gagné Stéphane M
PROTEO, the Québec Network for Research on Protein Structure, Function and Engineering, Université Laval, Québec, Canada.
Biomol NMR Assign. 2010 Oct;4(2):127-30. doi: 10.1007/s12104-010-9227-8. Epub 2010 Apr 10.
The rapid evolution of Class A β-lactamases, which procure resistance to an increasingly broad panel of β-lactam antibiotics, underscores the urgency to better understand the relation between their sequence variation and their structural and functional features. To date, more than 300 clinically-relevant β-lactamase variants have been reported, and this number continues to increase. With the aim of obtaining insights into the evolutionary potential of β-lactamases, an artificially engineered, catalytically active chimera of the Class A TEM-1 and PSE-4 β-lactamases is under study by kinetics and NMR. Here we report the (1)H, (13)C and (15)N backbone resonance assignments for the 30 kDa chimera cTEM-17m. Despite its high molecular weight, the data provide evidence that this artificially-evolved chimeric enzyme is well folded. The hydrolytic activity of cTEM-17m was determined using the chromogenic substrate CENTA, with K (M) = 160 ± 35 μM and k (cat) = 20 ± 4 s(-1), which is in the same range as the values for TEM-1 and PSE-4 β-lactamases.
A类β-内酰胺酶迅速进化，对越来越多的β-内酰胺抗生素产生耐药性，这凸显了更好地理解其序列变异与其结构和功能特征之间关系的紧迫性。迄今为止，已报道了300多种与临床相关的β-内酰胺酶变体，且这一数字还在持续增加。为了深入了解β-内酰胺酶的进化潜力，一种人工构建的、具有催化活性的A类TEM-1和PSE-4β-内酰胺酶嵌合体正在通过动力学和核磁共振进行研究。在此，我们报告了30 kDa嵌合体cTEM-17m的（1）H、（13）C和（15）N主链共振归属。尽管其分子量较大，但数据表明这种人工进化的嵌合酶折叠良好。使用显色底物CENTA测定了cTEM-17m的水解活性，K（M）= 160 ± 35 μM，k（cat）= 20 ± 4 s（-1），与TEM-1和PSE-4β-内酰胺酶的值处于同一范围。
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