Recombinant DNA-methyltransferase M1.Bst19I from Bacillus stearothermophilus 19: Purification, properties, and amino acid sequence analysis

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作者：
J. E. Tomilova
V. V. Kuznetsov
M. A. Abdurashitov
N. A. Netesova
S. Kh. Degtyarev
机构：
[1] SibEnzyme Novosibirsk,
[2] Vector State Research Center of Virology and Biotechnology,undefined
来源：
Molecular Biology | 2010年 / 44卷
关键词：
DNA-methyltransferases; enzyme kinetics; amino acid sequence;
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摘要：
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 Escherichia 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. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters of M1.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 (kcat) 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.
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页码：606 / 615
页数：9
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