A novel glycerol kinase from Flavobacterium meningosepticum: characterization, gene cloning and primary structure.
A thermostable glycerol kinase (FGK) was purified 34-fold to homogeneity from Flavobacterium meningosepticum. The molecular masses of the enzyme were 200 kDa by gel filtration and 50 kDa by SDS-PAGE. The Km for glycerol and ATP were 0.088 and 0.030 mM, respectively. The enzyme was stable at 65 degrees C for 10 min and at 37 degrees C for two weeks. The enzyme gene was cloned into Escherichia coli and its complete DNA was sequenced. The FGK gene consists of an open reading frame of 1494-bp encoding a protein of 498 amino acids. The deduced amino acid sequence of the gene had 40-60% similarity to those of glycerol kinases from other origins and the amino acid sequence of the putative active site residue reported for E. coli GK is identical to the corresponding sequence of FGK except for one amino acid residue.
Sakasegawa
S
S
Asahi Chemical Industry Co. Ltd., Shizuoka, Japan. a9310932@ut.asahi-kasei.co.jp
Yoshioka
I
I
Koga
S
S
Takahashi
M
M
Matsumoto
K
K
Misaki
H
H
Ohshima
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D002847:Chromatography, Agarose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005991:Glycerol Kinase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D005994:Glycerophosphates / Q000032:analysis; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity