Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis
Identification and Characterization of Two Adenosine Phosphorylase Activities in Mycobacterium smegmatis
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DOI：
10.1128/jb.05394-11
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发表时间：
2011-10-01
影响因子：
3.2
通讯作者：
Parker, William B.
中科院分区：
文献类型：
--
作者：
Buckoreelall, Kajal;Wilson, Landon;Parker, William B.
嘌呤核苷磷酸化酶（PNP）是嘌呤代谢中的重要酶，将嘌呤核苷切割成相应的碱基。分枝杆菌PNP对6-氧代嘌呤具有特异性，不能解释在分枝杆菌中检测到的腺苷（Ado）裂解活性。结核和M.粪污培养物。在目前的工作中，从M.包皮垢细胞提取物。第一种活性经生化测定为磷酸化酶，其可可逆地催化腺苷+磷酸腺嘌呤+ α-D-核糖-1-磷酸。我们的纯化方案导致该活性的30倍纯化，去除了超过99.9%的总蛋白。虽然Ado是优选的底物，但肌苷和鸟苷也被切割，分别具有43%和32%的Ado活性。我们的数据表明M. smeglobulin表达两种PNP：先前描述的仅能切割肌苷和鸟苷的三聚体PNP，以及第二种能切割腺苷、肌苷和鸟苷的新型PNP（p-PNP）。β-PNP的表观K-m（K-m（app））为98 +/- 6 μ M（含Ado），天然分子量为125 +/- 7 kDa。根据其生化特性和质谱分析，第二个Ado切割活性被鉴定为5 '-甲硫基腺苷磷酸化酶（MTAP）。我们的研究标志着MTAP在任何细菌中存在的第一份报告。由于人类细胞不容易将Ado转化为Ade，因此了解这些酶的底物偏好可能会导致识别出可以在分支杆菌中选择性激活为有毒产物的Ado类似物。
Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate adenine + alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K-m (K-m (app)) of 98 +/- 6 mu M (with Ado) and a native molecular mass of 125 +/- 7 kDa. The second Ado cleavage activity was identified as 5'-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.