Cloning, sequencing, high expression, and crystallization of the thermophile Thermus aquaticus glycerol kinase

• Published in: Bioscience, biotechnology, and biochemistry Vol. 62; no. 12; pp. 2375 - 2381
• Main Authors: Huang, H.S. (Nagasaki Univ. (Japan). Faculty of Pharmaceutical Sciences), Ito, K, Yin, C.H, Kabashima, T, Yoshimoto, T
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.12.1998; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: Amino Acid Sequence; Bacillus subtilis - enzymology; Bacillus subtilis - genetics; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; Blotting, Southern; Chromatography, Gel; CLONACION; CLONAGE; CLONING; Cloning, Molecular; CRISTALIZACION; CRISTALLISATION; CRYSTALLIZATION; DNA Primers - chemistry; DNA, Bacterial - chemistry; Escherichia coli - enzymology; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genetic engineering; Genetic technics; Genetics; GLICEROL; GLYCEROL; glycerol kinase; Glycerol Kinase - biosynthesis; Glycerol Kinase - chemistry; Glycerol Kinase - genetics; Glycerol Kinase - isolation & purification; Glycerophosphates - analysis; Hot Temperature; Methods. Procedures. Technologies; MICROORGANISME THERMOPHILE; MICROORGANISMOS TERMOFILOS; Mission oriented research; Modification of gene expression level; Molecular Sequence Data; Molecular Weight; NUCLEOTIDE SEQUENCE; Polymerase Chain Reaction; Recombinant Proteins - chemistry; SECUENCIA NUCLEOTIDICA; Sequence Analysis, DNA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Spectrophotometry; THERMOPHILIC MICROORGANISMS; thermostable enzyme; Thermus - enzymology; Thermus - genetics; Thermus aquaticus; TRANSFERASAS; TRANSFERASE; TRANSFERASES; Heterologous system; Nucleotide sequence; Thermus aquaticus; Enzyme; Transferases; Escherichia coli; Crystallization; Gene overexpression; Gene expression; Thermal stability; Gene; Glycerol kinase; Bacteria; Recombinant protein; Molecular cloning; Aminoacid sequence; Enterobacteriaceae; Structural analysis
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.62.2375

Glycerol kinase 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 than of the B. subtilis enzyme showed that Ser and Lys are replaced by Ala and Arg, as was seen in mesophile and thermophile enzymes