Ni Jinfeng, Sasaki Yoshito, Tokuyama Shinji, Sogabe Atsushi, Tahara Yasutaka
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
J Biosci Bioeng. 2002;93(1):31-6. doi: 10.1016/s1389-1723(02)80050-0.
We have converted a typical catalase from Bacillus sp. TE124 to a catalase-peroxidase using DNA shuffling and error-prone PCR. A triple mutant, R47H/R356C/D374N, that showed significantly reduced catalase activity and increased peroxidase activity was identified by screening mutant libraries. When single mutant--R47H, R356C and D374N--were generated by site-directed mutagenesis, conserved Arg-47, located on the distal side of the prosthetic heme group in the superfamily of typical catalases, was found to be responsible for the conversion of catalase to catalase-peroxidase. To further clarify the role of Arg-47, arginine was replaced with different amino acids--alanine, lysine, aspartic acid, glutamic acid, glutamine, phenylalanine, tryptophan and tyrosine--and the mutant enzymes were assayed. All of the arginine mutants had increased peroxidase activity coupled with reduced catalase activity. Among these mutants, R47W exhibited the highest peroxidase activity, while R47E and R47Q not only had increased peroxidase activity but also retained relatively high catalase activity. These results suggest that tryptophan plays a key role in the catalytic mechanism of the peroxidase reaction and that glutamic acid and glutamine facilitate both catalatic and peroxidatic reactions.
我们利用DNA改组和易错PCR技术，将一种来自芽孢杆菌属TE124的典型过氧化氢酶转化为过氧化氢酶-过氧化物酶。通过筛选突变体文库，鉴定出一个三突变体R47H/R356C/D374N，其过氧化氢酶活性显著降低而过氧化物酶活性增加。当通过定点诱变产生单突变体——R47H、R356C和D374N时，发现在典型过氧化氢酶超家族中位于辅基血红素基团远端的保守精氨酸-47，是过氧化氢酶转化为过氧化氢酶-过氧化物酶的原因。为了进一步阐明精氨酸-47的作用，将精氨酸替换为不同的氨基酸——丙氨酸、赖氨酸、天冬氨酸、谷氨酸、谷氨酰胺、苯丙氨酸、色氨酸和酪氨酸，并对突变酶进行检测。所有精氨酸突变体的过氧化物酶活性均增加，而过氧化氢酶活性降低。在这些突变体中，R47W表现出最高的过氧化物酶活性，而R47E和R47Q不仅过氧化物酶活性增加，而且还保留了相对较高的过氧化氢酶活性。这些结果表明，色氨酸在过氧化物酶反应的催化机制中起关键作用，而谷氨酸和谷氨酰胺促进催化反应和过氧化物反应。
