Increased Rigidity of Domain Structures Enhances the Stability of a Mutant Enzyme Created by Directed Evolution
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- 作者：Jun Hoseki ; Akihiro Okamoto ; Naoki Takada ; Atsushi Suenaga ; Noriyuki Futatsugi ; Akihiko Konagaya ; Makoto Taiji ; Takato Yano ; Seiki Kuramitsu ; and Hiroyuki Kagamiyama
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：December 16, 2003
- 年：2003
- 卷：42
- 期：49
- 页码：14469 - 14475
- 全文大小：500K
- 年卷期：v.42,no.49(December 16, 2003)
- ISSN：1520-4995
文摘
A mutant of kanamycin nucleotidyltransferase (KNT) was previously created by directedevolution. This mutant, HTK, has 19 amino acid substitutions, which increase the thermostability by 20
C. In this study, we have examined to what extent each mutation contributes to the increased stabilityand analyzed how the mutations affect the structure of KNT at 72 C using molecular dynamics simulations.The effects of some mutations on the stability are simply additive, but those of others are cooperative.Mutations with large effects on the stability are introduced into the N-terminal domain, which appears tobe less stable than the C-terminal domain. Results of the molecular dynamics simulations have indicatedthat the rigidity of the domain structures is increased by the mutations: at 72 C, the intradomain fluctuationsof HTK are decreased, and in turn, its interdomain motions are pronounced, whereas the structure of thepreevolved KNT fluctuates randomly. Chemical modification experiments of cysteine residues have shownthat the cysteine residues of HTK are less accessible to an SH reagent than those of the preevolved KNT.The present results suggest that the 19 mutations of HTK stabilize KNT by affecting the dynamic behaviorof the structure of this enzyme without significantly changing its static overall structure.
C. In this study, we have examined to what extent each mutation contributes to the increased stabilityand analyzed how the mutations affect the structure of KNT at 72 C using molecular dynamics simulations.The effects of some mutations on the stability are simply additive, but those of others are cooperative.Mutations with large effects on the stability are introduced into the N-terminal domain, which appears tobe less stable than the C-terminal domain. Results of the molecular dynamics simulations have indicatedthat the rigidity of the domain structures is increased by the mutations: at 72 C, the intradomain fluctuationsof HTK are decreased, and in turn, its interdomain motions are pronounced, whereas the structure of thepreevolved KNT fluctuates randomly. Chemical modification experiments of cysteine residues have shownthat the cysteine residues of HTK are less accessible to an SH reagent than those of the preevolved KNT.The present results suggest that the 19 mutations of HTK stabilize KNT by affecting the dynamic behaviorof the structure of this enzyme without significantly changing its static overall structure.