An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.
An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8.
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DOI：
10.1093/oxfordjournals.jbchem.a021198
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发表时间：
1996
影响因子：
2.7
通讯作者：
A. Okamoto;R. Kato;R. Masui;A. Yamagishi;Tairo Oshima;S. Kuramitsu
中科院分区：
文献类型：
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作者：
A. Okamoto;R. Kato;R. Masui;A. Yamagishi;Tairo Oshima;S. Kuramitsu
对一株极端嗜热菌Thermus thermophilus HB 8的天冬氨酸氨基转移酶基因（AspAT，EC 2.6.1.1）进行了克隆和序列测定，并对其基因产物进行了过量生产。纯化的T.嗜热菌AspAT在中性pH下稳定至约80 ℃。嗜热菌AspAT对酸性氨基酸底物如天冬氨酸、谷氨酸和相应的酮酸具有严格特异性。编码T.嗜热菌AspAT基因全长1,155 bp，G+C含量高（70 mol%），编码385个氨基酸残基，分子量为42050。T.从其基因推导的AspAT与大肠杆菌、芽孢杆菌YM-2和硫磺硫化叶菌的AspAT基因的同源性分别为15%、46%和29%。当T.嗜热菌AspAT与E. coli AspAT中，发现Cys的数目从5个减少到1个，Asn的数目从23个减少到9个，Gln的数目从16个减少到8个，Asp的数目从20个减少到13个，所有这些都是已知的在高温下相对不稳定的。相反，Pro的数量从15增加到25，Arg从22增加到32，Glu从27增加到37。如E. coliAspAT结构中，脯氨酰残基有明显的分布于分子表面的趋势。这与RecA蛋白的情况完全不同，RecA蛋白在其分子内部显示出脯氨酰残基数量的增加。不同蛋白质的脯氨酰对热稳定性的贡献不同的策略已被提出。尽管活性位点残基高度保守，但在E. coliAspAT与底物的末端羧基相互作用，而在T.嗜热菌Arg 89可能与Arg 292的功能互补。
The aspartate aminotransferase gene (AspAT, EC 2.6.1.1) of an extremely thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced, and its gene product was overproduced. The purified T. thermophilus AspAT was stable up to about 80 degrees C at neutral pH. T. thermophilus AspAT was strictly specific for acidic amino acid substrates, such as aspartate, glutamate, and the respective keto acids. The gene coding for T. thermophilus AspAT showed that it comprised 1,155 bp with a high G+C content (70 mol%), and encoded a 385-residue protein with a molecular weight of 42,050. The amino acid sequence of T. thermophilus AspAT deduced from its gene showed about 15, 46, and 29% homology with those from Escherichia coli, Bacillus sp. YM-2, and Sulfolobus solfataricus, respectively. When the amino acid sequence of T. thermophilus AspAT was compared with that of E. coli AspAT, the number of Cys was found to have decreased from 5 to 1, that of Asn from 23 to 9, that of Gln from 16 to 8, and that of Asp from 20 to 13, all of which are known to be relatively labile at high temperatures. Conversely, the number of Pro was increased from 15 to 25, Arg from 22 to 32, and Glu 27 to 37. As shown by the E. coli AspAT structure, there was a marked tendency for the extra prolyl residues to be located around the surface of the molecule. This was quite different from that in the case of RecA protein, which shows an increased number of prolyl residues in the interior of its molecule. Different strategies of different proteins as to prolyl contribution to thermostability have been suggested. Despite the high degree of conservation of active-site residues, Arg292 in E. coli AspAT, which interacts with the distal carboxylate of the substrate, was not found in T. thermophilus AspAT. Arg89 may complement the function of Arg292.