Okamoto A, Kato R, Masui R, Yamagishi A, Oshima T, Kuramitsu S
Department of Biology, Faculty of Science, Osaka University, Toyonaka.
J Biochem. 1996 Jan;119(1):135-44. doi: 10.1093/oxfordjournals.jbchem.a021198.
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.
克隆并测序了嗜热栖热菌HB8的天冬氨酸转氨酶基因（AspAT，EC 2.6.1.1），并过量表达了其基因产物。纯化后的嗜热栖热菌AspAT在中性pH条件下，温度高达约80℃时仍保持稳定。嗜热栖热菌AspAT对酸性氨基酸底物具有严格的特异性，如天冬氨酸、谷氨酸以及相应的酮酸。编码嗜热栖热菌AspAT的基因显示，其由1155个碱基对组成，G+C含量较高（70摩尔%），编码一个含有385个氨基酸残基、分子量为42050的蛋白质。从该基因推导得到的嗜热栖热菌AspAT的氨基酸序列与大肠杆菌、芽孢杆菌属YM - 2和嗜热栖硫叶菌的氨基酸序列分别具有约15%、46%和29%的同源性。当将嗜热栖热菌AspAT的氨基酸序列与大肠杆菌AspAT的氨基酸序列进行比较时，发现半胱氨酸的数量从5个减少到1个，天冬酰胺从23个减少到9个，谷氨酰胺从16个减少到8个，天冬氨酸从20个减少到13个，所有这些氨基酸在高温下都相对不稳定。相反，脯氨酸的数量从15个增加到25个，精氨酸从22个增加到32个，谷氨酸从27个增加到37个。如大肠杆菌AspAT结构所示，额外的脯氨酰残基明显倾向于位于分子表面。这与RecA蛋白的情况截然不同，RecA蛋白在其分子内部脯氨酰残基数量增加。已经提出了不同蛋白质关于脯氨酸对热稳定性贡献的不同策略。尽管活性位点残基具有高度保守性，但在嗜热栖热菌AspAT中未发现大肠杆菌AspAT中与底物远端羧酸盐相互作用的精氨酸292。精氨酸89可能补充了精氨酸292的功能。
