全球,结核病是第二大传染病杀手,多重耐药性严重阻碍了疾病的控制。霉菌酸是一种独特的脂质类别,对生存能力至关重要,毒力,以及病原体的持久性,结核分枝杆菌(Mtb)。因此,参与霉菌酸生物合成的酶代表了一类重要的药物靶标。我们先前表明,(3R)-羟酰基-ACP脱水酶(HAD)蛋白HadD主要用于生产酮霉素酸,并在Mtb生物膜形成和毒力中起决定性作用。这里,我们发现HAD活性需要在HadD和HadB之间形成紧密的异四聚体,由不同的染色体区域编码的HAD单元。利用生化,结构,和基于细胞的分析,我们发现HadB是催化亚基,而HadD参与底物结合。基于HadBDMtb晶体结构和底物结合模型,我们确定了超长链脂质底物特异性的决定因素,并揭示了结构-功能关系的细节。HadBDMtb的独特功能部分归因于HadD中底物结合缝隙的更宽开口和更高灵活性,以及HadD热狗褶皱的急剧截断的中央α螺旋,在HAD酶中首次描述的特征。一起来看,我们的研究表明HadBDMtb,而不是一个人,是生物学相关的功能单元。这些结果对设计创新的抗结核分子具有重要意义。因为他们认为要考虑的目标不是一个孤立的亚基,而是整个HadBD复合体.
Worldwide, tuberculosis is the second leading infectious killer and multidrug resistance severely hampers disease control. Mycolic acids are a unique category of lipids that are essential for viability, virulence, and persistence of the causative agent, Mycobacterium tuberculosis (Mtb). Therefore, enzymes involved in mycolic acid biosynthesis represent an important class of drug targets. We previously showed that the (3R)-hydroxyacyl-ACP dehydratase (HAD) protein HadD is dedicated mainly to the production of ketomycolic acids and plays a determinant role in Mtb biofilm formation and virulence. Here, we discovered that HAD activity requires the formation of a tight heterotetramer between HadD and HadB, a HAD unit encoded by a distinct chromosomal region. Using biochemical, structural, and cell-based analyses, we showed that HadB is the catalytic subunit, whereas HadD is involved in substrate binding. Based on HadBDMtb crystal structure and substrate-bound models, we identified determinants of the ultra-long-chain lipid substrate specificity and revealed details of structure-function relationship. HadBDMtb unique function is partly due to a wider opening and a higher flexibility of the substrate-binding crevice in HadD, as well as the drastically truncated central α-helix of HadD hotdog fold, a feature described for the first time in a HAD enzyme. Taken together, our study shows that HadBDMtb , and not HadD alone, is the biologically relevant functional unit. These results have important implications for designing innovative antivirulence molecules to fight tuberculosis, as they suggest that the target to consider is not an isolated subunit, but the whole HadBD complex.