Tokita K, Hori K, Kurotsu T, Kanda M, Saito Y
Department of Biochemistry, Hyogo College of Medicine.
J Biochem. 1993 Oct;114(4):522-7. doi: 10.1093/oxfordjournals.jbchem.a124210.
The mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: alpha-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthetase family.
从短芽孢杆菌长野BII - 3菌株中克隆并测序了编码脯氨酸激活结构域的突变基因（grs2 - pro），该菌株产生的短杆菌肽S合成酶2在脯氨酸激活方面存在缺陷。通过将核苷酸序列与野生型序列进行比较，发现在第2609位鸟嘌呤处有一个单点突变，该鸟嘌呤被腺嘌呤取代，导致第870位甘氨酸变为谷氨酸。对grs2 - pro基因推导的氨基酸序列进行同源性搜索发现，第870位甘氨酸在腺苷酸形成酶中保守，其侧翼序列在氨基酰腺苷酸形成酶中高度保守，如抗生素肽合成酶：短杆菌肽S合成酶1和2（GS1、GS2）、杀念菌素合成酶1（TS1）以及δ -（L -α -氨基己二酰基）-L -半胱氨酰 - D -缬氨酸合成酶（ACVS）；还有其他氨基酰腺苷酸化酶：α -氨基己二酸还原酶（LYS2）、EntF和AngR。另一方面，在缺乏氨基酸激活的其他腺苷酸形成酶中，如乙酰辅酶A合成酶、长链酰基辅酶A合成酶、荧光素酶和4 -香豆酸辅酶A连接酶，该侧翼序列不保守。通过寡核苷酸定点诱变对第870位GGG密码子进行单碱基替换。分离出四个诱变克隆，其包含将870 - Gly分别替换为丙氨酸、缬氨酸、精氨酸和色氨酸的grs2 - pro基因。这些克隆的翻译产物几乎不能催化脯氨酸依赖性ATP - 32PPi交换反应。突变体中870 - Gly区域的卷曲结构丧失。这些结果表明，grs2 - pro蛋白的870 - Gly残基对抗生素肽合成酶家族中的氨基酰腺苷酸化至关重要。
