Bashiri Ghader, Johnston Jodie M, Evans Genevieve L, Bulloch Esther M M, Goldstone David C, Jirgis Ehab N M, Kleinboelting Silke, Castell Alina, Ramsay Rochelle J, Manos-Turvey Alexandra, Payne Richard J, Lott J Shaun, Baker Edward N
School of Biological Sciences, University of Auckland, 3a Symonds Steet, Private Bag 90210, Auckland 1142, New Zealand.
School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.
Acta Crystallogr D Biol Crystallogr. 2015 Nov;71(Pt 11):2297-308. doi: 10.1107/S1399004715017216. Epub 2015 Oct 31.
The tryptophan-biosynthesis pathway is essential for Mycobacterium tuberculosis (Mtb) to cause disease, but not all of the enzymes that catalyse this pathway in this organism have been identified. The structure and function of the enzyme complex that catalyses the first committed step in the pathway, the anthranilate synthase (AS) complex, have been analysed. It is shown that the open reading frames Rv1609 (trpE) and Rv0013 (trpG) encode the chorismate-utilizing (AS-I) and glutamine amidotransferase (AS-II) subunits of the AS complex, respectively. Biochemical assays show that when these subunits are co-expressed a bifunctional AS complex is obtained. Crystallization trials on Mtb-AS unexpectedly gave crystals containing only AS-I, presumably owing to its selective crystallization from solutions containing a mixture of the AS complex and free AS-I. The three-dimensional structure reveals that Mtb-AS-I dimerizes via an interface that has not previously been seen in AS complexes. As is the case in other bacteria, it is demonstrated that Mtb-AS shows cooperative allosteric inhibition by tryptophan, which can be rationalized based on interactions at this interface. Comparative inhibition studies on Mtb-AS-I and related enzymes highlight the potential for single inhibitory compounds to target multiple chorismate-utilizing enzymes for TB drug discovery.
色氨酸生物合成途径对于结核分枝杆菌（Mtb）致病至关重要，但该生物体中催化此途径的所有酶尚未全部被鉴定出来。已经对催化该途径第一步即邻氨基苯甲酸合酶（AS）复合物的酶复合物的结构和功能进行了分析。结果表明，开放阅读框Rv1609（trpE）和Rv0013（trpG）分别编码AS复合物的分支酸利用（AS-I）和谷氨酰胺氨基转移酶（AS-II）亚基。生化分析表明，当这些亚基共表达时，可获得双功能AS复合物。对Mtb-AS进行的结晶试验意外地得到了仅含AS-I的晶体，推测这是由于它从含有AS复合物和游离AS-I混合物的溶液中选择性结晶所致。三维结构显示，Mtb-AS-I通过一个此前在AS复合物中未见的界面形成二聚体。与其他细菌的情况一样，已证明Mtb-AS受色氨酸的协同别构抑制作用，这可以基于该界面处的相互作用来解释。对Mtb-AS-I和相关酶的比较抑制研究突出了单一抑制性化合物针对多种分支酸利用酶用于结核病药物研发的潜力。
