Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria.
Expression of catalytic domains of human UMP synthase in uridine auxotrophic bacteria.
复制标题
人 UMP 合酶催化结构域在尿苷营养缺陷型细菌中的表达。
DOI：
10.1007/bf01233533
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Suttle,DP
中科院分区：
文献类型：
--
作者：
Lin,T;Suttle,DP
Orotate磷酸核糖基转移酶（OPRT）和orotidine-5 ' -单磷酸脱羧酶（ODC）是细菌和低等真核生物中两种不同的单功能蛋白，催化了UMP生物合成的最后两个步骤。在哺乳动物中，OPRT和ODC活性包含在一个标记为UMP合成酶的单一双功能蛋白中。利用聚合酶链反应技术将人UMP合成酶cDNA分离为预测的OPRT和ODC结构域，并将其插入pUC19表达载体中。在转化为缺乏OPRT和odc的大肠杆菌后，菌株能够在不含尿苷的最小培养基上生长。odc转化的细菌表达的活性水平是野生型大肠杆菌的24倍。oprt转化的大肠杆菌仅含有野生型活性的4-9%。人UMP合成酶抗血清的Western blot分析表明，OPRT和ODC结构域在缺陷细胞中由各自的载体产生。结构域蛋白的水平近似于酶活性的水平。在转化的缺陷大肠杆菌菌株中，OPRT和ODC活性的互补表明，人UMP合酶可以被分离成在细菌细胞环境中发挥作用的活性单功能结构域。
Orotate phosphoribosyltransferase (OPRT) and orotidine-5′-monophosphate decarboxylase (ODC), which catalyze the last two steps in de novo UMP biosynthesis, are two distinct monofunctional proteins in bacteria and lower eukaryotes. In mammals, OPRT and ODC activities are contained in a single bifunctional protein labeled UMP synthase. The human UMP synthase cDNA was separated into the predicted OPRT and ODC domains using polymerase chain reaction techniques and the domains inserted into pUC19 expression vectors. Following transformation into OPRT- and ODC-deficient E. coli, the strains were able to grow on minimal media without uridine. The ODC-transformed bacteria expressed up to 24 times the level of activity found in a wild-type E. coli line. The OPRT-transformed E. coli contained only 4–9% of wild-type activity. Western blot analysis with antiserum to human UMP synthase demonstrates that OPRT and ODC domains are being produced in the deficient cells by the respective vectors. The level of the domain protein approximates the level of enzyme activity. The complementation of the OPRT and ODC activities in the transformed deficient E. coli strains demonstrates that human UMP synthase can be separated into active monofunctional domains that will function in the bacterial cell environment.