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

Abstract

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 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 (k
cat) 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.

Key wordsDNA-methyltransferases-
Bacillus stearothermophilus
-enzyme kinetics-amino acid sequence