Namanja-Magliano Hilda A, Evans Gary B, Harijan Rajesh K, Tyler Peter C, Schramm Vern L
Department of Biochemistry, Albert Einstein College of Medicine , 1300 Morris Park Avenue, Bronx, New York 10461, United States.
The Ferrier Research Institute, Victoria University of Wellington , Lower Hutt, Wellington 5040, New Zealand.
Biochemistry. 2017 Sep 26;56(38):5090-5098. doi: 10.1021/acs.biochem.7b00576. Epub 2017 Sep 7.
Mycobacterium tuberculosis 5'-deoxyadenosine/5'-methylthioadenosine nucleosidase (Rv0091) catalyzes the N-riboside hydrolysis of its substrates 5'-methylthioadenosine (MTA) and 5'-deoxyadenosine (5'-dAdo). 5'-dAdo is the preferred substrate, a product of radical S-adenosylmethionine-dependent enzyme reactions. Rv0091 is characterized by a ribocation-like transition state, with low N-ribosidic bond order, an N7-protonated adenine leaving group, and an activated but weakly bonded water nucleophile. DADMe-Immucillins incorporating 5'-substituents of the substrates 5'-dAdo and MTA were synthesized and characterized as inhibitors of Rv0091. 5'-Deoxy-DADMe-Immucillin-A was the most potent among the 5'-dAdo transition state analogues with a dissociation constant of 640 pM. Among the 5'-thio substituents, hexylthio-DADMe-Immucillin-A was the best inhibitor at 87 pM. The specificity of Rv0091 for the Immucillin transition state analogues differs from those of other bacterial homologues because of an altered hydrophobic tunnel accepting the 5'-substituents. Inhibitors of Rv0091 had weak cell growth effects on M. tuberculosis or Mycobacterium smegmatis but were lethal toward Helicobacter pylori, where the 5'-methylthioadenosine nucleosidase is essential in menaquinone biosynthesis. We propose that Rv0091 plays a role in 5'-deoxyadenosine recycling but is not essential for growth in these Mycobacteria.
结核分枝杆菌5'-脱氧腺苷/5'-甲硫腺苷核苷酶（Rv0091）催化其底物5'-甲硫腺苷（MTA）和5'-脱氧腺苷（5'-dAdo）的N-核糖苷水解。5'-dAdo是首选底物，是自由基S-腺苷甲硫氨酸依赖性酶反应的产物。Rv0091的特征是具有类似核糖阳离子的过渡态，N-核糖苷键序低，N7-质子化腺嘌呤离去基团，以及活化但弱结合的水亲核试剂。合成了包含底物5'-dAdo和MTA的5'-取代基的DADMe-免疫亲和素，并将其表征为Rv0091的抑制剂。5'-脱氧-DADMe-免疫亲和素-A是5'-dAdo过渡态类似物中最有效的，解离常数为640 pM。在5'-硫代取代基中，己硫基-DADMe-免疫亲和素-A是最佳抑制剂，IC50为87 pM。由于接受5'-取代基的疏水通道发生改变，Rv0091对免疫亲和素过渡态类似物的特异性与其他细菌同源物不同。Rv0091的抑制剂对结核分枝杆菌或耻垢分枝杆菌的细胞生长影响较弱，但对幽门螺杆菌具有致死性，其中5'-甲硫腺苷核苷酶在甲萘醌生物合成中至关重要。我们认为Rv0091在5'-脱氧腺苷循环中起作用，但对这些分枝杆菌的生长不是必需的。
