Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB
Enzymological and structural studies of the mechanism of promiscuous substrate recognition by the oxidative DNA repair enzyme AlkB
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DOI：
10.1073/pnas.0812938106
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发表时间：
2009-08-25
影响因子：
11.1
通讯作者：
Hunt, John F.
中科院分区：
文献类型：
--
作者：
Yu, Bomina;Hunt, John F.
混杂的底物识别，催化化学上不同的化合物的转化的能力，是一种进化上有利的，但知之甚少的现象。DNA修复酶的混杂性特别重要，因为它使不同核苷酸碱基的不同类型的损伤能够以代谢简约的方式修复。我们目前的酶学和晶体学研究的机制混杂底物识别大肠杆菌AlkB，DNA修复酶，去除甲基加合物和一些较大的烷基化病变的内环位置上的嘌呤和嘧啶基地。体外Michaelis-Menten分析表明，一系列烷基化碱基在修复N1-甲基腺嘌呤（m1A）和N3-甲基胞嘧啶（m3 C）方面具有高活性，在修复1，N-6-乙烯基腺嘌呤方面活性相对较低，在修复N1-甲基鸟嘌呤或N3-甲基胸腺嘧啶方面没有可检测到的活性。与m1A相比，AlkB对于m3 C具有显著更高的k（cat）和K-m。因此，该酶通过增加具有名义上较低亲和力的底物的周转率来保持对化学上不同的底物的类似净活性。共晶结构提供了对这种“k（cat）/K-m补偿”的结构基础的深入了解，这对AlkB的混杂底物识别做出了重大贡献。在分析一个大的合奏的晶体结构解决在这些研究的过程中，我们观察到2个离散的全球构象AlkB不同的隧道假设控制扩散的O-2基板进入活性位点的可及性。一系列蛋白质环之间的空间相互作用控制这种构象转变，并提出了一个合理的机制，防止O-2结合之前，核苷酸底物结合。
Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1, N-6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher k(cat) and K-m for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this "k(cat)/K-m compensation,'' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O-2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O-2 binding before nucleotide substrate binding.