MmeI, a class-IIS restriction endonuclease: purification and characterization.
Two restriction endonucleases, MmeI and MmeII, from Methylophilus methylotrophus were purified to homogeneity. Both enzymes belong to the class-II restriction endonucleases (ENases) but exhibit very different enzymatic and physical properties. MmeII is a typical member of class-II ENases. It is a polymeric protein composed of 50-kDa subunits. In contrast to MmeII, MmeI is a monomeric protein of 101 kDa, cleaving a DNA molecule 20/18 nucleotides away from the asymmetric recognition sequence (5'-TCCRAC-3'); therefore, it is classified as a member of subclass-IIS. MmeI has an pI of 7.85 and is active in the pH range 6.5 to 10 with the optimum at 7 to 8. Increasing salt concentration creates an inhibitory effect on MmeI: 40 mM KCl decreases activity by 50%, 100 mM completely inhibits DNA cleavage. Tris.HCl (pH 7.5) at a concentration exceeding 20 mM inhibits MmeI activity. Mg2+ stimulates MmeI in the range of 0.2 to 35 mM, with the optimum between 0.5 and 10 mM.
Tucholski
J
J
Department of Microbiology, University of Gdańsk, Poland.
Skowron
P M
PM
Podhajska
A J
AJ
D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D015252:Deoxyribonucleases, Type II Site-Specific / Q000302:isolation & purification* / Q000378:metabolism*; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity
C049760:endodeoxyribonuclease MmeI; C094491:endodeoxyribonuclease MmeII; D015252:Deoxyribonucleases, Type II Site-Specific