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Vol 44(2010) N 4 p. 606-615; Y.E. Tomilova1*, V.V. Kusnetsov2, M.A. Abdurashitov1, N.A. Netesova2, S.K. Degtyarev1 Recombinant DNA-Methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: Purification, Properties, and Amino Acid Sequence Analysis 1SibEnzyme Research and Production Association, Novosibirsk, 630117 Russia2VECTOR State Research Center of Virology and Biotechnology, Kol'tsovo, Novosibirsk Region, 630559 Russia *JTom@sibenzyme.ru Received - 2009-12-29; Accepted - 2010-03-09 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 PR/PL from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from E. coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature optimum at 50°C and demonstrates maximal activity at pH 8.0. М1.Bst19I modifies adenine in sequence 5'-GCATC-3'. Kinetic parameters of М1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 + 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 + 0.31 μM. Catalytical constant (kсat) is 1.8 + 0.05 min-1. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence. DNA-methyltransferases, Bacillus stearothermophilus, enzyme kinetics, amino acid sequence |
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