Effect of mutations at Glu160 and Val198 on the thermostability of lactate oxidase.
We have obtained two types of thermostable mutant lactate oxidase - one that exhibited an E-to-G point mutation at position 160 (E160G) through error-prone PCR-based random mutagenesis, and another that exhibited an E-to-G mutation at position 160 and a V-to-I mutation at position 198 (E160G/V198I) through DNA shuffling-based random mutagenesis - both of which we have previously reported. Our molecular modeling of lactate oxidase suggests that the substitution of G for E at position 160 reduces the electrostatic repulsion between the negative charges of E160 and E130 in the (beta/alpha)8 barrel structure, but a thermal-inactivation experiment on the five kinds of single-mutant lactate oxidase at position 160 (E160A, E160Q, E160H, E160R, and E160K) showed that the side-chain volume of the amino acid at position 160 mainly contributes to the thermostability of lactate oxidase. We also produced V198I single-mutant lactate oxidase through site-directed mutagenesis, and analysed the thermostability of wild-type, V198I, E160G, and E160G/V198I lactate oxidase enzymes. The half-life of E160G/V198I lactate oxidase at 70 degrees C was about three times longer than that of E160G lactate oxidase, and was about 20 times longer than that of wild-type lactate oxidase. In contrast, the thermostability of the V198I lactate oxidase was almost identical to that of wild-type lactate oxidase. This indicates that the V198I mutation alone does not affect lactate oxidase thermostability, but does affect it when combined with the E160G mutation.
Minagawa
Hirotaka
H
Fundamental Research Laboratories, NEC Corp., Miyukigaoka, Tsukuba, Japan. h-minagawa@ab.jp.nec.com
Shimada
Jiro
J
Kaneko
Hiroki
H
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018724:Spinacia oleracea / Q000201:enzymology; D055672:Static Electricity; D013696:Temperature; D014633:Valine / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006899:Mixed Function Oxygenases; C021497:lactate 2-monooxygenase; D014633:Valine