Sabljić Igor, Meštrović Nevenka, Vukelić Bojana, Macheroux Peter, Gruber Karl, Luić Marija, Abramić Marija
Division of Physical Chemistry, Ruđer Bošković Institute, Zagreb, Croatia.
Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia.
PLoS One. 2017 Nov 2;12(11):e0187295. doi: 10.1371/journal.pone.0187295. eCollection 2017.
Bacteroides thetaiotaomicron is a dominant member of the human intestinal microbiome. The genome of this anaerobe encodes more than 100 proteolytic enzymes, the majority of which have not been characterized. In the present study, we have produced and purified recombinant dipeptidyl peptidase III (DPP III) from B. thetaiotaomicron for the purposes of biochemical and structural investigations. DPP III is a cytosolic zinc-metallopeptidase of the M49 family, involved in protein metabolism. The biochemical results for B. thetaiotaomicron DPP III from our research showed both some similarities to, as well as certain differences from, previously characterised yeast and human DPP III. The 3D-structure of B. thetaiotaomicron DPP III was determined by X-ray crystallography and revealed a two-domain protein. The ligand-free structure (refined to 2.4 Å) was in the open conformation, while in the presence of the hydroxamate inhibitor Tyr-Phe-NHOH, the closed form (refined to 3.3 Å) was observed. Compared to the closed form, the two domains of the open form are rotated away from each other by about 28 degrees. A comparison of the crystal structure of B. thetaiotaomicron DPP III with that of the human and yeast enzymes revealed a similar overall fold. However, a significant difference with functional implications was discovered in the upper domain, farther away from the catalytic centre. In addition, our data indicate that large protein flexibility might be conserved in the M49 family.
多形拟杆菌是人类肠道微生物群的主要成员。这种厌氧菌的基因组编码了100多种蛋白水解酶，其中大多数尚未得到表征。在本研究中，我们制备并纯化了来自多形拟杆菌的重组二肽基肽酶III（DPP III），用于生化和结构研究。DPP III是M49家族的一种胞质锌金属肽酶，参与蛋白质代谢。我们研究中多形拟杆菌DPP III的生化结果显示，它与先前表征的酵母和人类DPP III既有一些相似之处，也有某些差异。多形拟杆菌DPP III的三维结构通过X射线晶体学确定，显示为一种双结构域蛋白。无配体结构（精修至2.4 Å）处于开放构象，而在存在异羟肟酸酯抑制剂Tyr-Phe-NHOH的情况下，观察到封闭形式（精修至3.3 Å）。与封闭形式相比，开放形式的两个结构域彼此旋转了约28度。将多形拟杆菌DPP III的晶体结构与人类和酵母酶的晶体结构进行比较，发现总体折叠相似。然而，在远离催化中心的上部结构域中发现了一个具有功能意义的显著差异。此外，我们的数据表明，M49家族可能保留了较大的蛋白质灵活性。
