Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA
Crystallization and preliminary crystallographic analysis of the type IIL restriction enzyme MmeI in complex with DNA
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DOI：
10.1107/s1744309111028041
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发表时间：
2011-10-01
影响因子：
0.9
通讯作者：
Aggarwal, Aneel K.
中科院分区：
文献类型：
--
作者：
Callahan, Scott J.;Morgan, Richard D.;Aggarwal, Aneel K.
IIL型限制性内切酶使人们重新开始寻找用户指定的DNA结合和切割。通过比对和对比高度可比的氨基酸序列以及跨酶家族的不同识别特异性，已经鉴定并突变了参与DNA结合的氨基酸以产生替代结合特异性。迄今为止，MmeI（一种IIL型限制性内切酶）的特异性已经成功地在不对称TCCRAC（其中R是嘌呤）DNA识别序列的3、4和6位改变。为了进一步了解MmeI DNA结合特异性的结构基础，该酶已与其DNA底物复合结晶。该晶体属于空间群P1，晶胞参数a = 61.73，B = 94.96，c = 161.24埃，α = 72.79，β = 89.12，γ = 71.68度，当暴露于同步辐射时衍射至2.6埃分辨率。该结构有望揭示MmeI DNA结合特异性的基础，并将补充创造具有新特异性的酶的努力。
Type IIL restriction enzymes have rejuvenated the search for user-specified DNA binding and cutting. By aligning and contrasting the highly comparable amino-acid sequences yet diverse recognition specificities across the family of enzymes, amino acids involved in DNA binding have been identified and mutated to produce alternative binding specificities. To date, the specificity of MmeI (a type IIL restriction enzyme) has successfully been altered at positions 3, 4 and 6 of the asymmetric TCCRAC (where R is a purine) DNA-recognition sequence. To further understand the structural basis of MmeI DNA-binding specificity, the enzyme has been crystallized in complex with its DNA substrate. The crystal belonged to space group P1, with unit-cell parameters a = 61.73, b = 94.96, c = 161.24 angstrom, alpha = 72.79, beta = 89.12, gamma = 71.68 degrees, and diffracted to 2.6 angstrom resolution when exposed to synchrotron radiation. The structure promises to reveal the basis of MmeI DNA-binding specificity and will complement efforts to create enzymes with novel specificities.