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[摘要]：Adenine aminohydrolase (AAH) is an enzyme that is not present in mammalian cells and is found exclusively in Leishmania among the protozoan parasites that infect humans. AAH plays a paramount role in purine metabolism in this genus by steering 6-aminopurines into 6-oxypurines. Leishmania donovani AAH is 38 and 23% identical to Saccharomyces cerevisiae AAH and human adenosine deaminase enzymes, respectively, catalyzes adenine deamination to hypoxanthine with an apparent K-m of 15.4 mu M, and does not recognize adenosine as a substrate. Western blot analysis established that AAH is expressed in both life cycle stages of L. donovani, whereas subcellular fractionation and immunofluorescence studies confirmed that AAH is localized to the parasite cytosol. Deletion of the AAH locus in intact parasites established that AAH is not an essential gene and that Delta aah cells are capable of salvaging the same range of purine nucleobases and nucleosides as wild type L. donovani. The Delta aah null mutant was able to infect murine macrophages in vitro and in mice, although the parasite loads in both model systems were modestly reduced compared with wild type infections. The Delta aah lesion was also introduced into a conditionally lethal Delta hgprt/Delta xprt mutant in which viability was dependent on pharmacologic ablation of AAH by 2'-deoxycoformycin. The Delta aah/Delta hgprt/Delta xprt triple knock-out no longer required 2'-deoxycoformycin for growth and was avirulent in mice with no persistence after a 4-week infection. These genetic studies underscore the paramount importance of AAH to purine salvage by L. donovani. |
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