Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.
Staphylococcal phosphoenolpyruvate-dependent phosphotransferase system: purification and characterization of the mannitol-specific enzyme IIImtl of Staphylococcus aureus and Staphylococcus carnosus and homology with the enzyme IImtl of Escherichia coli.
复制标题
葡萄球菌磷酸烯醇丙酮酸依赖性磷酸转移酶系统：金黄色葡萄球菌和肉葡萄球菌甘露醇特异性酶 IIImtl 的纯化和表征以及与大肠杆菌酶 IImtl 的同源性。
作者：
B. Reiche;R. Frank;J. Deutscher;N. Meyer;W. Hengstenberg
IIImtl酶是金黄色葡萄球菌和肉毒葡萄球菌甘露醇磷酸转移酶系统的一部分，被磷酸烯醇丙酮酸磷酸化，其反应顺序需要酶I（磷酸烯醇丙酮酸-蛋白磷酸转移酶）和含组氨酸的蛋白HPr。本文报道了金黄色葡萄球菌（S. aureus）和肉毒杆菌（S. carnosus）中IIImtl的分离及活性中心的鉴定。在用[32P]PEP、酶I和HPr磷酸化IIImtl后，磷酸化蛋白用内源性蛋白酶Glu(C)裂解。结果表明，携带该磷酸基的金黄色葡萄球菌肽的氨基酸序列为Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp。鼠耳草中相应的肽序列相同，只是第一个残基是Ala而不是Gln。这些多肽都含有一个单一的组氨酸残基，我们假设它携带磷基。迄今为止研究的所有PTS蛋白确实携带与组氨酸残基相连的磷酸化基团。根据十二烷基硫酸钠凝胶，发现IIImtl蛋白的分子量为15,000。我们还确定了这两种蛋白的n端序列。将IImtl肽序列与大肠杆菌IImtl酶的c端序列进行比较，发现其序列具有相当的同源性，支持大肠杆菌IImtl是可溶性III蛋白与膜结合酶II的融合蛋白的观点。特别是，金黄色葡萄球菌和肉毒杆菌的活性中心肽与大肠杆菌的酶IImtl的同源性使人们能够预测大肠杆菌酶内的N-3组氨酸磷酸化位点。
Enzyme IIImtl is part of the mannitol phosphotransferase system of Staphylococcus aureus and Staphylococcus carnosus and is phosphorylated by phosphoenolpyruvate in a reaction sequence requiring enzyme I (phosphoenolpyruvate-protein phosphotransferase) and the histidine-containing protein HPr. In this paper, we report the isolation of IIImtl from both S. aureus and S. carnosus and the characterization of the active center. After phosphorylation of IIImtl with [32P]PEP, enzyme I, and HPr, the phosphorylated protein was cleaved with endoproteinase Glu(C). The amino acid sequence of the S. aureus peptide carrying the phosphoryl group was found to be Gln-Val-Val-Ser-Thr-Phe-Met-Gly-Asn-Gly-Leu-Ala-Ile-Pro-His-Gly-Thr-Asp- Asp. The corresponding peptide from S. carnosus shows an equal sequence except that the first residue is Ala instead of Gln. These peptides both contain a single histidyl residue which we assume to carry the phosphoryl group. All proteins of the PTS so far investigated indeed carry the phosphoryl group attached to a histidyl residue. According to sodium dodecyl sulfate gels, the molecular weight of the IIImtl proteins was found to be 15,000. We have also determined the N-terminal sequence of both proteins. Comparison of the IIImtl peptide sequences and the C-terminal part of the enzyme IImtl of Escherichia coli reveals considerable sequence homology, which supports the suggestion that IImtl of E. coli is a fusion protein of a soluble III protein with a membrane-bound enzyme II. In particular, the homology of the active-center peptide of IIImtl of S. aureus and S. carnosus with the enzyme IImtl of E. coli allows one to predict the N-3 histidine phosphorylation site within the E. coli enzyme.