Goulding Celia W, Apostol Marcin I, Sawaya Michael R, Phillips Martin, Parseghian Angineh, Eisenberg David
Molecular Biology Institute UCLA-DOE Institute of Genomics and Proteomics, P.O. Box 951570, Los Angeles, CA 90095-1570, USA.
J Mol Biol. 2005 May 27;349(1):61-72. doi: 10.1016/j.jmb.2005.03.023. Epub 2005 Apr 2.
Folate derivatives are essential cofactors in the biosynthesis of purines, pyrimidines and amino acids across all forms of life. Mammals uptake folate from their diets, whereas most bacteria must synthesize folate de novo. Therefore, the enzymes in the folate biosynthetic pathway are attractive drug targets against bacterial pathogens such as Mycobacterium tuberculosis, the cause of the world's most deadly infectious disease, tuberculosis (TB). M.tuberculosis 7,8-dihydroneopterin aldolase (Mtb FolB, DHNA) is the second enzyme in the folate biosynthetic pathway, which catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and glycoaldehyde. The 1.6A X-ray crystal structure of Mtb FolB complexed with its product, 6-hydroxymethyl-7,8-dihydropterin, reveals an octameric assembly similar to that seen in crystal structures of other FolB homologs. However, the 2.5A crystal structure of unliganded Mtb FolB reveals a novel tetrameric oligomerization state, with only partially formed active sites. A substrate induced conformational change appears to be necessary to convert the inactive tetramer to the active octamer. Ultracentrifugation confirmed that in solution unliganded Mtb FolB is mainly tetrameric and upon addition of substrate FolB is predominantly octameric. Kinetic analysis of substrate binding gives a Hill coefficient of 2.0, indicating positive cooperativity. We hypothesize that Mtb FolB displays cooperativity in substrate binding to regulate the cellular concentration of 7,8-dihydroneopterin, so that it may function not only as a precursor to folate but also as an antioxidant for the survival of M.tuberculosis against host defenses.
叶酸衍生物是所有生命形式中嘌呤、嘧啶和氨基酸生物合成过程中必不可少的辅助因子。哺乳动物从饮食中摄取叶酸，而大多数细菌必须从头合成叶酸。因此，叶酸生物合成途径中的酶是针对结核分枝杆菌等细菌病原体的有吸引力的药物靶点，结核分枝杆菌是世界上最致命的传染病——结核病（TB）的病原体。结核分枝杆菌7,8-二氢新蝶呤醛缩酶（Mtb FolB，DHNA）是叶酸生物合成途径中的第二种酶，它催化7,8-二氢新蝶呤转化为6-羟甲基-7,8-二氢蝶呤和乙醇醛。与产物6-羟甲基-7,8-二氢蝶呤复合的Mtb FolB的1.6埃X射线晶体结构显示出一种八聚体组装，类似于在其他FolB同源物的晶体结构中看到的组装。然而，未结合配体的Mtb FolB的2.5埃晶体结构揭示了一种新的四聚体寡聚化状态，只有部分形成的活性位点。底物诱导的构象变化似乎是将无活性的四聚体转化为有活性的八聚体所必需的。超速离心证实，在溶液中未结合配体的Mtb FolB主要是四聚体，加入底物后FolB主要是八聚体。底物结合的动力学分析给出的希尔系数为2.0，表明存在正协同性。我们假设Mtb FolB在底物结合中表现出协同性，以调节7,8-二氢新蝶呤的细胞浓度，从而使其不仅可以作为叶酸的前体，还可以作为结核分枝杆菌对抗宿主防御生存的抗氧化剂发挥作用。
