[Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: purification, propeties and amino acids sequence analysis].
The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5'-GCATC-3') in Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW, that carries a tandem of thermo inducible promoters P(R)/P(L) from phage lambda. Highly purified enzyme has been isolated by chromatography on various resins from E. coli cells where it accumulates in a soluble form. Study of M1.Bst19I properties has revealed that enzyme has a temperature optimum 50 degrees C and demonstrates the maximum activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5'-GCATC-3'. Kinetic parameters of M1.Bst19I DNA methylation reaction have been determined as follows: Km for lambda DNA is 0.68 +/- 0.07 microM, Km for S-adenosil-L-methionine is 2.02 +/- 0.31 microM. Catalytical constant (kcat) is 1.8 +/- 0.05 min(-1). Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and others alpha-N6-DNA methyltransferases has allowed to suppose a presence of two types of the enzymes containing a triplet ATG or ATC in the recognition sequence.
Tomilova
Iu É
IuÉ
Kuznetsov
V V
VV
Abdurashitov
M A
MA
Netesova
N A
NA
Degtiarev
S Kh
SKh
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D010582:Bacteriophage lambda / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000737:chemistry* / Q000378:metabolism; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D004279:DNA, Viral; D011994:Recombinant Proteins; D015254:DNA Modification Methylases