Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit

• Published in: FEMS microbiology letters Vol. 270; no. 1; pp. 171 - 177
• Main Authors: Cajthamlová, Kamila, Šišáková, Eva, Weiser, Jaroslav, Weiserová, Marie
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01-05-2007; Blackwell; Oxford University Press
• Subjects: Bacteriology; Biological and medical sciences; Blotting, Western; Chromatography, Thin Layer; Deoxyribonucleases, Type I Site-Specific - metabolism; DNA Restriction Enzymes - metabolism; E coli; Electrophoresis; Endonuclease; Enzymes; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - metabolism; Escherichia coli Proteins - metabolism; Fundamental and applied biological sciences. Psychology; Immunoblotting; Immunoprecipitation; Kinases; Metabolism. Enzymes; Microbiology; Phosphoamino Acids - metabolism; Phosphoproteins; Phosphorylation; Protein kinase; protein phosphorylation; Protein Subunits - metabolism; Restriction-modification; subcellular fraction; restriction-modification; subcellular fraction; protein phosphorylation; Restriction modification; Phosphorylation; Enzyme; restriction- modification; Hydrolases; Esterases; Subunit; Protein; Endodeoxyribonuclease EcoKI
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.2007.00663.x

Abstract
Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I – representatives of IA, IB, and IC families, respectively – was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits – as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.