Watanabe S, Muramatsu T, Ao H, Hirayama Y, Takahashi K, Tanokura M, Kuchino Y
Biophysics Division, National Cancer Center Research Institute, Tokyo, Japan.
Eur J Biochem. 1999 Dec;266(3):811-9. doi: 10.1046/j.1432-1327.1999.00907.x.
The gene encoding Lon protease was isolated from an extreme thermophile, Thermus thermophilus HB8. Sequence analysis demonstrated that the T. thermophilus Lon protease gene (TT-lon) contains a protein-coding sequence consisting of 2385 bp which is approximately 56% homologous to the Escherichia coli counterpart. As expected, the G/C content of TT-lon was 68%, which is significantly higher than that of the E. coli lon gene (52% G/C). The amino acid sequence of T. thermophilus Lon protease (TT-Lon) predicted from the nucleotide sequence contained several unique sequences conserved in other Lon proteases: (a) a cysteine residue at the position just before the putative ATP-binding domain; (b) motif A and B sequences required for composition of the ATP-binding domain; and (c) a serine residue at the proteolytic active site. Expression of TT-lon under the control of the T7 promoter in E. coli produced an 89-kDa protein with a yield of approximately 5 mg.L-1. Recombinant TT-Lon (rTT-Lon) was purified to homogeneity by sequential column chromatography. The peptidase activity of rTT-Lon was activated by ATP and alpha-casein. rTT-Lon cleaved succinyl-phenylalanyl-leucyl-phenylalanyl-methoxynaphthylamide much more efficiently than succinyl-alanyl-alanyl-phenylalanyl-methoxynaphthylamide, whereas both peptides were cleaved with comparable efficiencies by E. coli Lon. These results suggest that there is a difference between TT-Lon and E. coli Lon in substrate specificity. rTT-Lon most effectively cleaved substrate peptides at 70 degrees C, which was significantly higher than the optimal temperature (37 degrees C) for E. coli Lon. Together, these results indicate that the TT-lon gene isolated from T. thermophilus HB8 actually encodes an ATP-dependent thermostable protease Lon.
编码Lon蛋白酶的基因是从嗜热栖热菌HB8中分离得到的。序列分析表明，嗜热栖热菌Lon蛋白酶基因（TT-lon）包含一个由2385 bp组成的蛋白质编码序列，该序列与大肠杆菌的对应序列具有约56%的同源性。正如预期的那样，TT-lon的G/C含量为68%，显著高于大肠杆菌lon基因的G/C含量（52%）。从核苷酸序列预测的嗜热栖热菌Lon蛋白酶（TT-Lon）的氨基酸序列包含几个在其他Lon蛋白酶中保守的独特序列：（a）在假定的ATP结合结构域之前位置的一个半胱氨酸残基；（b）组成ATP结合结构域所需的基序A和B序列；（c）蛋白水解活性位点的一个丝氨酸残基。在大肠杆菌中，TT-lon在T7启动子的控制下表达产生了一种89 kDa的蛋白质，产量约为5 mg·L-1。重组TT-Lon（rTT-Lon）通过连续柱层析纯化至同质。rTT-Lon的肽酶活性被ATP和α-酪蛋白激活。rTT-Lon切割琥珀酰-苯丙氨酰-亮氨酰-苯丙氨酰-甲氧基萘酰胺的效率比琥珀酰-丙氨酰-丙氨酰-苯丙氨酰-甲氧基萘酰胺高得多，而大肠杆菌Lon对这两种肽的切割效率相当。这些结果表明，TT-Lon和大肠杆菌Lon在底物特异性上存在差异。rTT-Lon在70℃时最有效地切割底物肽，这显著高于大肠杆菌Lon的最佳温度（37℃）。总之这些结果表明，从嗜热栖热菌HB8中分离得到的TT-lon基因实际上编码一种依赖ATP的耐热蛋白酶Lon。
