Johansson Patrik, Castell Alina, Jones T Alwyn, Bäckbro Kristina
Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, SE-751 24 Uppsala, Sweden.
Protein Sci. 2006 Oct;15(10):2300-9. doi: 10.1110/ps.062309306. Epub 2006 Sep 8.
A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k(cat) of 1.1 x 10(2) sec(-1). The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.
结核分枝杆菌基因组的很大一部分编码功能未知的蛋白质。我们在此报告其中一种蛋白质Rv0130的结构,其分辨率为1.8埃。Rv0130单体具有单个热狗折叠结构,由一个高度弯曲的β折叠片层位于一个长α螺旋和一个短α螺旋之上组成。两个单体进而堆积形成一个双热狗折叠的同型二聚体,类似于一大类以硫酯为底物的酶。发现Rv0130含有一个高度保守的R特异性水合酶基序,深深埋藏在两个单体之间。我们的生化研究表明,该蛋白质能够使一个短的反式-2-烯酰辅酶A部分水合,催化常数k(cat)为1.1×10²秒⁻¹。定点诱变证明了D40和H45侧链对水合酶活性的重要性。与许多热狗折叠蛋白不同,一个脯氨酸残基扭曲了Rv0130的中央螺旋。这种扭曲允许形成一个长的、弯曲的通道,类似于长链真核水合酶2酶的底物结合通道。