The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme.
The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.
Leclere
M M
MM
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Japan.
Nishioka
M
M
Yuasa
T
T
Fujiwara
S
S
Takagi
M
M
Imanaka
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D004260:DNA Repair; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000235:genetics*; D010802:Phylogeny; D019711:Pyrococcus / Q000201:enzymology*; D011995:Recombination, Genetic
D019853:O(6)-Methylguanine-DNA Methyltransferase