Comparative studies of the phage T2 and T4 DNA (N6-adenine)methyltransferases: amino acid changes that affect catalytic activity.
The bacteriophage T2 and T4 dam genes code for a DNA (N6-adenine)methyltransferase (MTase). Nonglucosylated, hydroxymethylcytosine-containing T2gt- virion DNA has a higher level of methylation than T4gt- virion DNA does. To investigate the basis for this difference, we compared the intracellular enzyme levels following phage infection as well as the in vitro intrinsic methylation capabilities of purified T2 and T4 Dam MTases. Results from Western blotting (immunoblotting) showed that the same amounts of MTase protein were produced after infection with T2 and T4. Kinetic analyses with purified homogeneous enzymes showed that the two MTases had similar Km values for the methyl donor, S-adenosyl-L-methionine, and for substrate DNA. In contrast, they had different k(cat) values (twofold higher for T2 Dam MTase). We suggest that this difference can account for the ability of T2 Dam to methylate viral DNA in vivo to a higher level than does T4 Dam. Since the T2 and T4 MTases differ at only three amino acid residues (at positions 20 [T4, Ser; T2, Pro], 26 [T4, Asn; T2, Asp], and 188 [T4, Asp; T2, Glu]), we have produced hybrid proteins to determine which residue(s) is responsible for increased catalytic activity. The results of these analyses showed that the residues at positions 20 and 26 are responsible for the different k(cat) values of the two MTases for both canonical and noncanonical sites. Moreover, a single substitution of either residue 20 or 26 was sufficient to increase the k(cat) of T4 Dam.
Kossykh
V G
VG
Department of Biology, University of Rochester, New York 14627, USA.
Schlagman
S L
SL
Hattman
S
S
D000596:Amino Acids / Q000235:genetics*; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D002384:Catalysis; D019175:DNA Methylation; D004279:DNA, Viral / Q000032:analysis*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017900:Myoviridae / Q000201:enzymology* / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D000596:Amino Acids; D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C499296:dam protein, Bacteriophage T2; C499297:dam protein, Bacteriophage T4