Cheng Yu-Shan, Sacchettini James C
Department of Chemistry, Texas A&M University , College Station, Texas 77842, United States.
Department of Biochemistry and Biophysics, Texas A&M University , College Station, Texas 77843, United States.
Biochemistry. 2016 Feb 23;55(7):1107-19. doi: 10.1021/acs.biochem.5b00993. Epub 2016 Feb 5.
Mycobacterium tuberculosis (Mtb) Rv2671 is annotated as a 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (AROPP) reductase (RibD) in the riboflavin biosynthetic pathway. Recently, a strain of Mtb with a mutation in the 5' untranslated region of Rv2671, which resulted in its overexpression, was found to be resistant to dihydrofolate reductase (DHFR) inhibitors including the anti-Mtb drug para-aminosalicylic acid (PAS). In this study, a biochemical analysis of Rv2671 showed that it was able to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolate (THF), which explained why the overexpression of Rv2671 was sufficient to confer PAS resistance. We solved the structure of Rv2671 in complex with the NADP(+) and tetrahydrofolate (THF), which revealed the structural basis for the DHFR activity. The structures of Rv2671 complexed with two DHFR inhibitors, trimethoprim and trimetrexate, provided additional details of the substrate binding pocket and elucidated the differences between their inhibitory activities. Finally, Rv2671 was unable to catalyze the reduction of AROPP, which indicated that Rv2671 and its closely related orthologues are not involved in riboflavin biosynthesis.
结核分枝杆菌（Mtb）的Rv2671在核黄素生物合成途径中被注释为5-氨基-6-核糖基氨基-2,4（1H，3H）-嘧啶二酮5'-磷酸（AROPP）还原酶（RibD）。最近，发现一株Rv2671的5'非翻译区发生突变导致其过表达的结核分枝杆菌对包括抗结核药物对氨基水杨酸（PAS）在内的二氢叶酸还原酶（DHFR）抑制剂具有抗性。在本研究中，对Rv2671的生化分析表明，它能够催化二氢叶酸（DHF）还原为四氢叶酸（THF），这解释了Rv2671的过表达足以赋予PAS抗性的原因。我们解析了Rv2671与NADP(+)和四氢叶酸（THF）复合物的结构，揭示了DHFR活性的结构基础。Rv2671与两种DHFR抑制剂甲氧苄啶和三甲曲沙复合物的结构提供了底物结合口袋的更多细节，并阐明了它们抑制活性之间的差异。最后，Rv2671无法催化AROPP的还原，这表明Rv2671及其密切相关的直系同源物不参与核黄素的生物合成。
