Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase.
Glycerol kinase (EC 2.7.1.30) is a key enzyme of glycerol uptake and metabolism in bacteria. Using PCR, we amplified and cloned a glycerol kinase gene, glpK, from Thermus aquaticus. The complete gene has 1488 base pairs, coding for a protein of 496 amino acids with a predicted molecular weight of 54,814. The amino acid sequence deduced from T. aquaticus glpK was found to have identities of 97 and 81%, respectively, with those of Thermus flavus and Bacillus subtilis glpK genes. After overproduction in Escherichia coli, the expressed enzyme was easily purified to homogeneity by DEAE-Toyopearl chromatography. The purified enzyme has been crystallized by the hanging drop vapor diffusion method at 22 degrees C. Comparison of the amino acid sequence with that of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes.
Huang
H S
HS
School of Pharmaceutical Sciences, Nagasaki University, Japan.
Ito
K
K
Yin
C H
CH
Kabashima
T
T
Yoshimoto
T
T
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D015964:Gene Expression Regulation, Bacterial; D005991:Glycerol Kinase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D005994:Glycerophosphates / Q000032:analysis; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013824:Thermus / Q000201:enzymology* / Q000235:genetics