Hatanaka Tadashi, Negishi Tomofumi, Mori Koichi
Research Institute for Biological Sciences, Okayama, 7549-1 Kayo-cho, Jyobo-gun, Okayama 716-1241, Japan.
Biochim Biophys Acta. 2004 Jan 14;1696(1):75-82. doi: 10.1016/j.bbapap.2003.09.013.
To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.
为了研究氨基酸残基对磷脂酶D（PLD）热稳定性的贡献，构建了两种链霉菌PLD（热不稳定的K1PLD和热稳定的TH - 2PLD）的嵌合形式。K/T/KPLD是将K1PLD的329 - 441位残基与TH - 2PLD的同源区域重组而成，其热稳定性介于K1PLD和TH - 2PLD之间。通过比较链霉菌PLD的一级结构，确定了K1PLD中与热稳定性相关的七个氨基酸残基候选位点。通过定点诱变将K1PLD的Glu346替换为Asp得到的K1E346DPLD突变体表现出增强的热稳定性，几乎与TH - 2PLD相同。
