Increased rigidity of domain structures enhances the stability of a mutant enzyme created by directed evolution.
A mutant of kanamycin nucleotidyltransferase (KNT) was previously created by directed evolution. This mutant, HTK, has 19 amino acid substitutions, which increase the thermostability by 20 degrees C. In this study, we have examined to what extent each mutation contributes to the increased stability and analyzed how the mutations affect the structure of KNT at 72 degrees 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 to be less stable than the C-terminal domain. Results of the molecular dynamics simulations have indicated that the rigidity of the domain structures is increased by the mutations: at 72 degrees C, the intradomain fluctuations of HTK are decreased, and in turn, its interdomain motions are pronounced, whereas the structure of the preevolved KNT fluctuates randomly. Chemical modification experiments of cysteine residues have shown that 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 behavior of the structure of this enzyme without significantly changing its static overall structure.
Hoseki
Jun
J
Department of Biochemistry, Osaka Medical College, Takatsuki, Japan.
Okamoto
Akihiro
A
Takada
Naoki
N
Suenaga
Atsushi
A
Futatsugi
Noriyuki
N
Konagaya
Akihiko
A
Taiji
Makoto
M
Yano
Takato
T
Kuramitsu
Seiki
S
Kagamiyama
Hiroyuki
H
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution / Q000379:methods*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004795:Enzyme Stability / Q000235:genetics; D006358:Hot Temperature; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D013439:Sulfhydryl Reagents / Q000737:chemistry; D013816:Thermodynamics
D000596:Amino Acids; D001426:Bacterial Proteins; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D009713:Nucleotidyltransferases; C024251:kanamycin nucleotidyltransferase; D003545:Cysteine; D000409:Alanine