Rational design of thermostable lactate oxidase by analyzing quaternary structure and prevention of deamidation.
Our current knowledge of protein unfolding is overwhelmingly related to reversible denaturation. However, to engineer thermostable enzymes for industrial applications and medical diagnostics, it is necessary to consider irreversible denaturation processes and/or the entire quaternary structure. In this study we have used lactate oxidase (LOD), which is employed in lactic acid sensors, as a model example to design thermostable variants by rational design. Twelve mutant proteins were tested and one of them displayed a markedly greater thermostability than all the mutants we had previously obtained by random mutagenesis. This mutant was designed so as to strengthen the interaction between the subunits and stabilize the quaternary structure. Since LOD is difficult to crystallize, its three-dimensional structure remains unknown. This study shows that it is possible to carry out rational design to improve thermostability using a computer-aided quaternary structure model based on the known tertiary structure of a related protein. Critical factors required for increasing the thermal stability of proteins by rational design, where the 3-D structure is not available, are discussed.
Kaneko
Hiroki
H
Department of Integrated Sciences in Physics and Biology, College of Humanities and Sciences, Nihon University, 156-8550, Tokyo, Setagaya, Japan. kaneko@phys.chs.nihon-u.ac.jp
Minagawa
Hirotaka
H
Shimada
Jiro
J
D000577:Amides / Q000737:chemistry*; D019943:Amino Acid Substitution; D003198:Computer Simulation; D017076:Computer-Aided Design; D015195:Drug Design; D004795:Enzyme Stability; D024861:Genetic Enhancement / Q000379:methods; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D020836:Protein Structure, Quaternary; D013289:Streptococcaceae / Q000201:enzymology / Q000235:genetics; D013329:Structure-Activity Relationship
D000577:Amides; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase