Zhang W, Li L, Jiang W, Zhao G, Yang Y, Chiao J
Department of Microbiology, Shanghai Institute of Plant Physiology, Academia Sinica, Shanghai, P.R. China.
Eur J Biochem. 2000 Jun;267(12):3744-52. doi: 10.1046/j.1432-1327.2000.01410.x.
Genomic DNA sequencing in the vicinity of methylmalonyl-CoA mutase gene (mutAB) from a rifamycin SV-producing Amycolatopsis mediterranei U32 allowed us to clone, sequence, and identify a gene encoding a novel serine/threonine protein kinase (amk). The sequence contains a complete ORF of 1821 base pairs encoding a predicted protein of 606 amino acids in length. The N-terminal domain of the protein shows significant homology to the catalytic domain of other protein kinases from both prokaryotic and eukaryotic sources. It also contains all the structural features that are highly conserved in active protein kinases, including the Gly-X-Gly-X-X-Gly motif of ATP-binding and the essential amino acids known to be important for the recognition of the correct hydroxyamino acid in serine/threonine protein kinase. This protein kinase gene was expressed in Escherichia coli and was shown to have the ability of autophosphorylation. The autophosphorylated site was found to be the threonine at position 164 by labeled phosphoamino acid analysis and site-directed mutagenesis. The C-terminal half of protein kinase was found to contain strong transmembrane structures by PhoA fusion protein analysis, suggesting that Amk protein kinase is a transmembrane protein. A Southern hybridization experiment showed that this type of protein kinase is distributed ubiquitously and might play significant physiological roles in the various species of streptomycetes. However, overexpression of amk gene in Streptomyces cinnamonensis showed no effect on methylmalonyl-CoA mutase activity, monensin production and the hyphae morphology. Although its biological role is still unknown, Amk protein kinase is the first transmembrane serine/threonine protein kinase described for genus Amycolatopsis.
对利福霉素SV产生菌地中海拟无枝酸菌U32的甲基丙二酰辅酶A变位酶基因（mutAB）附近区域进行基因组DNA测序，使我们能够克隆、测序并鉴定一个编码新型丝氨酸/苏氨酸蛋白激酶（amk）的基因。该序列包含一个1821个碱基对的完整开放阅读框，编码一个预测长度为606个氨基酸的蛋白质。该蛋白质的N端结构域与来自原核和真核生物的其他蛋白激酶的催化结构域具有显著同源性。它还包含在活性蛋白激酶中高度保守的所有结构特征，包括ATP结合的Gly-X-Gly-X-X-Gly基序以及已知对丝氨酸/苏氨酸蛋白激酶中正确羟基氨基酸识别很重要的必需氨基酸。该蛋白激酶基因在大肠杆菌中表达，并显示具有自磷酸化能力。通过标记的磷酸氨基酸分析和定点诱变发现自磷酸化位点是第164位的苏氨酸。通过PhoA融合蛋白分析发现蛋白激酶的C端一半含有强跨膜结构，表明Amk蛋白激酶是一种跨膜蛋白。Southern杂交实验表明，这种类型的蛋白激酶广泛分布，可能在各种链霉菌物种中发挥重要的生理作用。然而，在肉桂链霉菌中过表达amk基因对甲基丙二酰辅酶A变位酶活性、莫能菌素产量和菌丝形态没有影响。尽管其生物学作用仍然未知，但Amk蛋白激酶是为拟无枝酸菌属描述的第一个跨膜丝氨酸/苏氨酸蛋白激酶。
