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Abstract：
AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive \'oxyferryl\' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein\'s Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.
摘要：
AlkB是Fe（II）/α-酮戊二酸依赖性双加氧酶，可修复大肠杆菌中DNA和RNA的某些烷基化碱基。在催化过程中，共底物(α-酮戊二酸，αKG）导致形成高度反应性的“氧铁蛋白”酶结合中间体，Fe(IV)=O,确保烷基核碱基加合物的羟基化。先前的研究表明，AlkB是一种灵活的蛋白质，在与辅因子和DNA相互作用时可以采用不同的构象。为了以实时模式评估与单链或双链DNA复合的酶的构象动力学，我们采用了停流荧光法。选择引入15聚体寡核苷酸序列中的N1-甲基腺嘌呤(m1A)作为特异性损伤。通过蛋白质Trp残基的固有荧光监测单周转动力学，荧光碱基类似物2-氨基嘌呤（2aPu），和染料-猝灭剂对(FAM/BHQ1)。对于所有的荧光标记,荧光痕迹显示了几个阶段一致的构象变化，被分配到酶促过程的具体步骤。这些数据提供了AlkB和DNA相互作用过程中结构动力学的总体图景。
