Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region.
The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was investigated. A single Escherichia coli transformant, which produced a more thermostable glycerol kinase than the parent enzyme, was obtained. The nucleotide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostability and Km for ATP had changed. The thermostability of FGK2615 was apparently increased; the temperature at which the enzyme activity is inactivated by 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degrees C which was 3.1 degrees C higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermostability and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.
Sakasegawa
S
S
Asahi Kasei Corporation, Shizuoka 410-2321, Japan. sakasegawa.sb@om.asahi-kasei.co.jp
Takehara
H
H
Yoshioka
I
I
Takahashi
M
M
Kagimoto
Y
Y
Misaki
H
H
Sakuraba
H
H
Ohshima
T
T
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004795:Enzyme Stability / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005991:Glycerol Kinase / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D016296:Mutagenesis; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity; D013997:Time Factors
D011994:Recombinant Proteins; D001224:Aspartic Acid; D012694:Serine; D000255:Adenosine Triphosphate; D005991:Glycerol Kinase