The Two Deubiquitinating Enzymes from Chlamydia trachomatis Have Distinct Ubiquitin Recognition Properties.
The Two Deubiquitinating Enzymes from Chlamydia trachomatis Have Distinct Ubiquitin Recognition Properties.
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DOI：
10.1021/acs.biochem.9b01107
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发表时间：
2020-04-28
期刊：
影响因子：
2.9
通讯作者：
Das C
中科院分区：
文献类型：
--
作者：
Hausman JM;Kenny S;Iyer S;Babar A;Qiu J;Fu J;Luo ZQ;Das C
沙眼衣原体是全球范围内多种疾病的病原体，可引发性传播的泌尿生殖系统疾病或眼部沙眼。该病原体基因组较小，但在感染时会表达两种具有去泛素化活性的酶，分别称为ChlaDUB1和ChlaDUB2。由于二者催化结构域的一级结构相似，推测它们具有冗余的去泛素酶（DUB）功能。此前的研究已对ChlaDUB1的酶学性质进行了结构表征，但ChlaDUB2尚未得到深入研究。在本研究中，我们对ChlaDUB2的去泛素酶性质展开研究，并将其与ChlaDUB1进行比较。结果显示，在水解二聚和多聚泛素链方面，二者的水解活性存在明显差异，然而在切割基于单泛素的底物泛素氨基甲基香豆素（Ub - AMC）时，二者表现出相似的能力。ChlaDUB2无法高效切割二聚泛素底物，而ChlaDUB1能够像典型的原核去泛素酶SdeA一样快速水解该底物。对于多聚泛素化的绿色荧光蛋白底物（GFP - Ubn），ChlaDUB1能有效地将多聚泛素链分解为单泛素产物，而ChlaDUB2虽使底物减少，但并未产生明显水平的单泛素产物。我们报道了ChlaDUB2催化结构域及其与泛素炔丙基酰胺复合物的结构。这些结构揭示了两种衣原体去泛素酶在底物识别相关残基上的差异。基于这些结构，我们得出结论：两种去泛素酶在远端泛素结合方面相当，这与Ub - AMC活性结果一致。因此，在较长泛素化底物上的活性差异，可能是由于对这些底物的识别不同，涉及额外的泛素结合位点。
Chlamydia trachomatis is the cause of several diseases worldwide in the form of a sexually transmitted urogenital disease or ocular trachoma. The pathogen contains a small genome yet, upon infection, expresses two enzymes with deubiquitinating activity, termed ChlaDUB1 and ChlaDUB2, presumed to have redundant deubiquitinase (DUB) function on account of similarity of primary structure of their catalytic domain. Previous studies have led to structural characterization of enzymatic properties of ChlaDUB1 however, ChlaDUB2 has yet to be investigated thoroughly. In this study, we investigated the deubiquitinase properties of ChlaDUB2 and compared them to that of ChlaDUB1. This revealed a distinct difference in hydrolytic activity with regards to di- and polyubiquitin chains, while showing similar ability to cleave a monoubiquitin-based substrate, ubiquitin aminomethylcoumarin (Ub-AMC). ChlaDUB2 was unable to cleave a diubiquitin substrate efficiently whereas ChlaDUB1 could rapidly hydrolyze this substrate comparable to a prototypical prokaryotic DUB, SdeA. With polyubiquitinated green fluorescent protein substrate (GFP-Ubn), whereas ChlaDUB1 efficiently disassembled the polyubiquitin chains into monoubiquitin product, the deubiquitination activity of ChlaDUB2, while showing a depletion of the substrate, did not produce appreciable levels of the monoubiquitin product. We report the structures of a catalytic construct of ChlaDUB2 and its complex with ubiquitin propargyl amide. These structures revealed differences in residues involved in substrate recognition between the two Chlamydia DUBs. Based on the structures we conclude that the distal ubiquitin binding is equivalent between the two DUBs, consistent with the Ub-AMC activity result. Therefore, the difference in activity with longer ubiquitinated substrates may be due to differential recognition of these substrates involving additional ubiquitin binding sites.
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