In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region.
Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival.
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
M
M
Ishida
Y
Y
Kanamori
N
N
Hori
T
T
Inaoka
E
E
Ohtsuka
D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D045647:DNA Glycosylases; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D005809:Genes, Regulator; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000235:genetics*; D010957:Plasmids; D013728:Terminator Regions, Genetic; D014466:Ultraviolet Rays; D014764:Viral Proteins
D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C036401:deoxyribopyrimidine endonucleosidase