Department of Pharmacology and Toxicology, University of Alabama at Birmingham, 1530 Third Avenue South, Birmingham, Alabama 35294, USA.
J Bacteriol. 2011 Oct;193(20):5668-74. doi: 10.1128/JB.05394-11. Epub 2011 Aug 5.
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 μM (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.
嘌呤核苷磷酸化酶（PNP）是嘌呤代谢中的一种重要酶，可将嘌呤核苷分解为各自的碱基。分枝杆菌 PNP 特异性作用于 6-氧嘌呤，不能解释在结核分枝杆菌和耻垢分枝杆菌培养物中检测到的腺苷（Ado）裂解活性。在当前的工作中，从耻垢分枝杆菌细胞提取物中鉴定出两种 Ado 裂解活性。第一种活性在生化上被确定为一种磷酸化酶，可可逆地催化腺苷+磷酸↔腺嘌呤+α-D-核糖-1-磷酸。我们的纯化方案使该活性的纯化度提高了 30 倍，去除了超过 99.9%的总蛋白。虽然 Ado 是首选底物，但肌苷和鸟苷也被裂解，分别占 Ado 活性的 43%和 32%。我们的数据表明，耻垢分枝杆菌表达两种 PNP：一种先前描述的三聚体 PNP，只能裂解肌苷和鸟苷，另一种新型 PNP（Ado-PNP），可裂解 Ado、肌苷和鸟苷。Ado-PNP 的表观 K(m)（K(m)（app））为 98±6μM（用 Ado 表示），天然分子量为 125±7kDa。第二种 Ado 裂解活性被鉴定为 5'-甲基硫代腺苷磷酸化酶（MTAP），基于其生化特性和质谱分析。我们的研究首次报道了 MTAP 在任何细菌中的存在。由于人类细胞不易将 Ado 转化为 Ade，因此了解这些酶的底物偏好性可能会导致鉴定出可在分枝杆菌中选择性激活为毒性产物的 Ado 类似物。
