Pioszak Augen A, Ninfa Alexander J
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
J Bacteriol. 2003 Feb;185(4):1299-315. doi: 10.1128/JB.185.4.1299-1315.2003.
Mutant forms of Escherichia coli NRII (NtrB) were isolated that retained wild-type NRII kinase activity but were defective in the PII-activated phosphatase activity of NRII. Mutant strains were selected as mimicking the phenotype of a strain (strain BK) that lacks both of the related PII and GlnK signal transduction proteins and thus has no mechanism for activation of the NRII phosphatase activity. The selection and screening procedure resulted in the isolation of numerous mutants that phenotypically resembled strain BK to various extents. Mutations mapped to the glnL (ntrB) gene encoding NRII and were obtained in all three domains of NRII. Two distinct regions of the C-terminal, ATP-binding domain were identified by clusters of mutations. One cluster, including the Y302N mutation, altered a lid that sits over the ATP-binding site of NRII. The other cluster, including the S227R mutation, defined a small surface on the "back" or opposite side of this domain. The S227R and Y302N proteins were purified, along with the A129T (NRII2302) protein, which has reduced phosphatase activity due to a mutation in the central domain of NRII, and the L16R protein, which has a mutation in the N-terminal domain of NRII. The S227R, Y302N, and L16R proteins were specifically defective in the PII-activated phosphatase activity of NRII. Wild-type NRII, Y302N, A129T, and L16R proteins bound to PII, while the S227R protein was defective in binding PII. This suggests that the PII-binding site maps to the "back" of the C-terminal domain and that mutation of the ATP-lid, central domain, and N-terminal domain altered functions necessary for the phosphatase activity after PII binding.
分离出了大肠杆菌NRII（NtrB）的突变形式，这些突变体保留了野生型NRII激酶活性，但在PII激活的NRII磷酸酶活性方面存在缺陷。选择突变菌株以模拟一种菌株（BK菌株）的表型，该菌株同时缺乏相关的PII和GlnK信号转导蛋白，因此没有激活NRII磷酸酶活性的机制。通过选择和筛选程序，分离出了许多在表型上不同程度类似于BK菌株的突变体。突变定位到编码NRII的glnL（ntrB）基因，并且在NRII的所有三个结构域中都获得了突变。通过突变簇鉴定出C末端ATP结合结构域的两个不同区域。一个簇，包括Y302N突变，改变了位于NRII的ATP结合位点上方的一个盖子。另一个簇，包括S227R突变，确定了该结构域“背面”或相对侧的一个小表面。纯化了S227R和Y302N蛋白，以及A129T（NRII2302）蛋白，该蛋白由于NRII中央结构域的突变而磷酸酶活性降低，还有L16R蛋白，该蛋白在NRII的N末端结构域有突变。S227R、Y302N和L16R蛋白在NRII的PII激活的磷酸酶活性方面存在特异性缺陷。野生型NRII、Y302N、A129T和L16R蛋白与PII结合，而S227R蛋白在结合PII方面存在缺陷。这表明PII结合位点定位于C末端结构域的“背面”，并且ATP盖子、中央结构域和N末端结构域的突变改变了PII结合后磷酸酶活性所需的功能。
