[Cloning, expression and characterization of the hypoxanthine-guanine phosphoribosyltransferase mutants from T. tengcongensis].
Based on a predicted three-dimensional structure of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) from Thermoanaerobacter tengcongensis, three mutants of HGPRT were designed to modify the purine specificity of HGPRT. Site-directed mutagenesis was used to generate the three mutants, K(133)A, K(133)S, K(133)T. Wild type HGPRT and its mutants were expressed E. coli in BL21 (DE3) pLysS, and the expression products of them reached about 30% of the total protein. The molecular weight of the recombinant proteins was 22 kD. The specific activities of the enzymes were determined. Catalytic activities of mutants K(133)A, K(133)S, K(133)T retained only 4%, 1.1%, 2.7% activities, respectively, of the wild type for hypoxanthine, 1.7%   0.6%   1% activities, respectively, of wild type for guanine. However, the three mutants showed 24-fold, 7-fold, 18-fold activities, respectively, of the wild type for xanthine, and 650-fold, 210-fold, 380-fold activities, respectively, of the wild type for adenine. Comparison of kinetic data for purified recombinant mutant with wild-type HGPRT showed significant difference in the catalytic efficiency (kcat/Km) for purine, xanthine and adenine, the mutants exhibiting more than 40 to 50-fold higher kcat/Km, as a result of nearly 4 to 5-fold decrease in Km, compared with wild-type. These results demonstrate that a single amino acid substitution in HGPRT at the active site can significantly modify the specificity for binding purine.
You
De-Lin
DL
Stake Key Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing, China.
Qu
Hong
H
Chen
Qiang
Q
Xing
Yang
Y
Gu
Xiao-Cheng
XC
Luo
Ming
M
D000595:Amino Acid Sequence; D001421:Bacteria, Anaerobic / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007041:Hypoxanthine Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011994:Recombinant Proteins; D007041:Hypoxanthine Phosphoribosyltransferase