Lyons Bronwyn, Ravulapalli Ravikiran, Lanoue Jason, Lugo Miguel R, Dutta Debajyoti, Carlin Stephanie, Merrill A Rod
From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and.
the Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada.
J Biol Chem. 2016 May 20;291(21):11198-215. doi: 10.1074/jbc.M115.707653. Epub 2016 Mar 21.
A bioinformatics strategy was used to identify Scabin, a novel DNA-targeting enzyme from the plant pathogen 87.22 strain of Streptomyces scabies Scabin shares nearly 40% sequence identity with the Pierisin family of mono-ADP-ribosyltransferase toxins. Scabin was purified to homogeneity as a 22-kDa single-domain enzyme and was shown to possess high NAD(+)-glycohydrolase (Km (NAD) = 68 ± 3 μm; kcat = 94 ± 2 min(-1)) activity with an RSQXE motif; it was also shown to target deoxyguanosine and showed sigmoidal enzyme kinetics (K0.5(deoxyguanosine) = 302 ± 12 μm; kcat = 14 min(-1)). Mass spectrometry analysis revealed that Scabin labels the exocyclic amino group on guanine bases in either single-stranded or double-stranded DNA. Several small molecule inhibitors were identified, and the most potent compounds were found to inhibit the enzyme activity with Ki values ranging from 3 to 24 μm PJ34, a well known inhibitor of poly-ADP-ribosyltransferases, was shown to be the most potent inhibitor of Scabin. Scabin was crystallized, representing the first structure of a DNA-targeting mono-ADP-ribosyltransferase enzyme; the structures of the apo-form (1.45 Å) and with two inhibitors (P6-E, 1.4 Å; PJ34, 1.6 Å) were solved. These x-ray structures are also the first high resolution structures of the Pierisin subgroup of the mono-ADP-ribosyltransferase toxin family. A model of Scabin with its DNA substrate is also proposed.
采用生物信息学策略鉴定出一种来自植物病原菌疮痂链霉菌87.22菌株的新型DNA靶向酶Scabin。Scabin与单ADP核糖基转移酶毒素的Pierisin家族具有近40%的序列同一性。Scabin被纯化至同质，为一种22 kDa的单结构域酶，显示具有高NAD(+) - 糖水解酶活性（Km (NAD) = 68 ± 3 μM；kcat = 94 ± 2 min(-1)），带有RSQXE基序；还显示其靶向脱氧鸟苷并呈现S形酶动力学（K0.5(脱氧鸟苷) = 302 ± 12 μM；kcat = 14 min(-1)）。质谱分析表明，Scabin标记单链或双链DNA中鸟嘌呤碱基上的环外氨基。鉴定出几种小分子抑制剂，发现最有效的化合物抑制酶活性的Ki值范围为3至24 μM。PJ34是一种众所周知的多ADP核糖基转移酶抑制剂，被证明是Scabin最有效的抑制剂。Scabin被结晶，代表了一种DNA靶向单ADP核糖基转移酶的首个结构；解析了无配体形式（1.45 Å）以及与两种抑制剂（P6 - E，1.4 Å；PJ34，1.6 Å）结合的结构。这些X射线结构也是单ADP核糖基转移酶毒素家族Pierisin亚组的首个高分辨率结构。还提出了Scabin与其DNA底物的模型。
