Bradbeer C, Gudmundsdottir A
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
J Bacteriol. 1990 Sep;172(9):4919-26. doi: 10.1128/jb.172.9.4919-4926.1990.
The binding of calcium and cobalamin to outer membranes from cells of Escherichia coli that contained amplified levels of wild-type or mutant btuB was studied. The mutant (BBam50) had an aspartyl-prolyl dipeptide inserted after the original 50th amino acid residue of the mature BtuB protein, which is within a region that shows extensive homology with the ferric siderophore receptors. This insertion resulted in cleavage of the BtuB in two places. The larger form retained the insertion but had lost 11 amino acid residues from the amino terminus. The smaller form was cut at the insertion site. Both the wild-type protein and the larger form of mutant BtuB showed calcium-dependent cobalamin binding with the same affinity for cobalamin, although the mutant had a much lower affinity for calcium. The smaller form of the mutant BtuB protein had a greatly reduced affinity for cobalamin, which was probably the result of inactivation of the cobalamin-dependent calcium-binding site. Cobalamin-dependent calcium binding was measured in wild-type BtuB preparations and was found to have the same corrinoid specificity and response to various corrinoid concentrations as shown previously for cobalamin binding. The results are consistent with a role for calcium in the cobalamin pump of the outer membrane of E. coli and show that a conserved part of the BtuB protein is required for the cobalamin-dependent binding of calcium.
研究了钙和钴胺素与含有野生型或突变型btuB扩增水平的大肠杆菌细胞外膜的结合情况。突变体（BBam50）在成熟BtuB蛋白的第50个氨基酸残基之后插入了一个天冬氨酰-脯氨酰二肽，该区域与铁载体受体具有广泛的同源性。这种插入导致BtuB在两个位置被切割。较大的形式保留了插入部分，但从氨基末端失去了11个氨基酸残基。较小的形式在插入位点被切割。野生型蛋白和突变型BtuB的较大形式都显示出钙依赖性钴胺素结合，对钴胺素的亲和力相同，尽管突变体对钙的亲和力要低得多。突变型BtuB蛋白的较小形式对钴胺素的亲和力大大降低，这可能是钴胺素依赖性钙结合位点失活的结果。在野生型BtuB制剂中测量了钴胺素依赖性钙结合，发现其具有与先前钴胺素结合所示相同的类咕啉特异性和对各种类咕啉浓度的反应。结果与钙在大肠杆菌外膜钴胺素泵中的作用一致，并表明BtuB蛋白的一个保守部分是钴胺素依赖性钙结合所必需的。
