Chai Ai-Fen, Bulloch Esther M M, Evans Genevieve L, Lott J Shaun, Baker Edward N, Johnston Jodie M
Laboratory of Structural Biology, School of Biological Sciences and Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
Acta Crystallogr D Biol Crystallogr. 2015 Apr;71(Pt 4):862-72. doi: 10.1107/S1399004715001650. Epub 2015 Mar 26.
Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated with Mtb virulence. Here, the crystal structure of an Mtb enzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14-21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusual cis double bond in the mycobactin acyl chain.
结核分枝杆菌（Mtb）是结核病的病原体。在宿主巨噬细胞中获取铁依赖于称为分枝杆菌素的铁螯合铁载体，并且与Mtb毒力密切相关。在此，以2.30Å的分辨率展示了参与分枝杆菌素生物合成的Mtb酶MbtN与其FAD辅因子形成的复合物的晶体结构。MbtN的多肽折叠符合酰基辅酶A脱氢酶（ACAD）家族的折叠，与其将双键引入分枝杆菌素酰基链的预测作用一致。结构比较以及同一基因位点中酰基载体蛋白MbtL的存在表明，MbtN作用于酰基 - （酰基载体蛋白）而非酰基辅酶A。晶体结构的一个显著特征是从FAD的N(5)伸出的管状密度。这被解释为共价结合的聚乙二醇（PEG）片段，位于底物酰基可能结合的疏水口袋中。该口袋可容纳14 - 21个碳原子的酰基链，与分枝杆菌素酰基链的预期长度一致。支持这一点的是，稳态动力学表明MbtN具有ACAD活性，偏好至少16个碳原子的酰基链。相对于其他已进行结构表征的ACAD，酰基结合口袋（相对于FAD）采用不同的取向。这种差异可能与MbtN催化分枝杆菌素酰基链中形成不寻常的顺式双键的明显能力相关。
