Swarbrick Crystall M D, Bythrow Glennon V, Aragao David, Germain Gabrielle A, Quadri Luis E N, Forwood Jade K
School of Biomedical Sciences, Charles Sturt University , Wagga Wagga, New South Wales 2678, Australia.
Department of Biology, Brooklyn College, City University of New York , 2900 Bedford Avenue, Brooklyn, New York 11210, United States.
Biochemistry. 2017 Mar 14;56(10):1460-1472. doi: 10.1021/acs.biochem.6b01049. Epub 2017 Mar 2.
Mycobacteria contain a large number of highly divergent species and exhibit unusual lipid metabolism profiles, believed to play important roles in immune invasion. Thioesterases modulate lipid metabolism through the hydrolysis of activated fatty-acyl CoAs; multiple copies are present in mycobacteria, yet many remain uncharacterized. Here, we undertake a comprehensive structural and functional analysis of a TesB thioesterase from Mycobacterium avium (MaTesB). Structural superposition with other TesB thioesterases reveals that the Asp active site residue, highly conserved across a wide range of TesB thioesterases, is mutated to Ala. Consistent with these structural data, the wild-type enzyme failed to hydrolyze an extensive range of acyl-CoA substrates. Mutation of this residue to an active Asp residue restored activity against a range of medium-chain length fatty-acyl CoA substrates. Interestingly, this Ala mutation is highly conserved across a wide range of Mycobacterium species but not found in any other bacteria or organism. Our structural homology analysis revealed that at least one other TesB acyl-CoA thioesterase also contains an Ala residue at the active site, while two other Mycobacterium TesB thioesterases harbor an Asp residue at the active site. The inactive TesBs display a common quaternary structure that is distinct from that of the active TesB thioesterases. Investigation of the effect of expression of either the catalytically active or inactive MaTesB in Mycobacterium smegmatis exposed, to the best of our knowledge, the first genotype-phenotype association implicating a mycobacterial tesB gene. This is the first report that mycobacteria encode active and inactive forms of thioesterases, the latter of which appear to be unique to mycobacteria.
分枝杆菌包含大量高度分化的物种，并展现出不同寻常的脂质代谢特征，据信这些特征在免疫侵袭中发挥重要作用。硫酯酶通过水解活化的脂肪酰辅酶A来调节脂质代谢；分枝杆菌中存在多个拷贝，但许多仍未被鉴定。在此，我们对鸟分枝杆菌的一种TesB硫酯酶（MaTesB）进行了全面的结构和功能分析。与其他TesB硫酯酶的结构叠加显示，在广泛的TesB硫酯酶中高度保守的天冬氨酸活性位点残基突变为丙氨酸。与这些结构数据一致，野生型酶无法水解多种酰基辅酶A底物。将该残基突变为活性天冬氨酸残基可恢复对一系列中链长度脂肪酰辅酶A底物的活性。有趣的是，这种丙氨酸突变在广泛的分枝杆菌物种中高度保守，但在任何其他细菌或生物体中均未发现。我们的结构同源性分析表明，至少另一种TesB酰基辅酶A硫酯酶在活性位点也含有一个丙氨酸残基，而另外两种分枝杆菌TesB硫酯酶在活性位点含有一个天冬氨酸残基。无活性的TesB呈现出一种与活性TesB硫酯酶不同的共同四级结构。据我们所知，在耻垢分枝杆菌中对催化活性或无活性的MaTesB表达的影响进行研究，揭示了第一个涉及分枝杆菌tesB基因的基因型-表型关联。这是首次报道分枝杆菌编码硫酯酶的活性和无活性形式，其中无活性形式似乎是分枝杆菌所特有的。
