Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain.
Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain.
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猪葡萄球菌、金黄色葡萄球菌和杂种脂肪酶的比较生化和分子分析。
DOI：
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Friedrich Götz
中科院分区：
文献类型：
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作者：
Klaus Nikoleit;R. Rosenstein;Hubertus M. Verheij;Friedrich Götz
将来自金黄色葡萄球菌NCTC 8530的脂肪酶基因geh克隆到肉葡萄球菌中。DNA序列分析显示，该基因的开放阅读框（ORF）为2046个核苷酸，编码682个氨基酸，分子量为76900 Da，转录起始位点的确定显示了一个203个核苷酸的mRNA前导序列。在蛋白酶阴性的S.在pT 181 copSA 22的培养上清中发现了83-kDa脂肪酶的过表达。N-末端蛋白质测序和序列比较与其他三个葡萄球菌脂肪酶表明，这种脂肪酶被组织为前原酶。该脂肪酶的底物特异性不同于猪葡萄球菌脂肪酶。色葡萄hyicus脂肪酶表达高钙依赖性磷脂酶和脂肪酶活性，而S.金黄色葡萄球菌脂肪酶缺乏这种磷脂酶活性，并且其与三丁酰甘油或对硝基苯基辛酸酯的活性几乎不受Ca 2+离子的刺激。构建了杂合蛋白，其中S. hyicus脂肪酶C端145个残基被S.金黄色葡萄球菌脂肪酶，其含有建议的活性位点氨基酸Asp 602和His 641。杂交酶仍然是活跃的，并揭示了中间酶活性。最显著的影响是它失去了S。hyicus特异性磷脂酶的活性和，在对比的两个亲本酶，其活性与对硝基苯基辛酸变得高度敏感的存在下的Ca 2+。这些观察结果表明，S。Hyicus脂肪酶对磷脂的极性头基的结合袋有很大贡献。Ca（2+）结合位点可能位于S.猪脂肪酶两种密切相关的酶对Ca 2+的需求不同，这一事实强调了它起着结构作用而不是催化作用的概念。
The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
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作者：
P. Matsudaira
通讯作者：
P. Matsudaira