Studies of a DNA modification methyltransferase from Rhodobacter sphaeroides

The DNA methyltransferases represent an interesting class of enzymes for the study of protein-DNA interactions due to their high specificity, structural simplicity, and biological importance. RsrI methyltransferase (M$\cdot$RsrI) from Rhodobacter sphaeroides recognizes duplex d(GAATTC) and deposits methyl groups at the $N\sp6$ position of central adenine. M$\cdot$RsrI was purified to homogeneity from R. sphaeroides, and its gene cloned and sequenced. The purification used four chromatography columns, and yielded up to 100 $\mu$g of enzyme. M$\cdot$RsrI was overexpressed in E. coli and the yield of the purification improved by about 100-fold with respect to that from R. sphaeroides.Several physical and biochemical properties of the RsrI methyltransferase were determined: molecular weight under denaturing and nondenaturing conditions, isoelectric point, optimal reaction conditions, the mode of methyl group transfer, and the enzyme-DNA binding. M$\cdot$RsrI was compared to a functionally identical enzyme from E. coli, EcoRI methyltransferase (M$\cdot$EcoRI). M$\cdot$RsrI and M$\cdot$EcoRI differ in their physical properties, including a striking lack of similarity in their deduced amino acid sequences. The two methyltransferases might recognize the cognate DNA sequence differently, because they do not bind to DNA with equal efficiencies under the same conditions. However, M$\cdot$RsrI and M$\cdot$EcoRI share identical catalytic properties. Both transfer one methyl group to the recognition sequence per binding event.M$\cdot$RsrI and M$\cdot$EcoRI represent an opportunity to elucidate the mode of action of two structurally different but functionally identical enzymes. It is possible that these enzymes retain functional similarity by having essentially the same three dimensional configuration. Alternatively, they might have dissimilar structures as well as mechanistic differences.U of I OnlyETDs are only available to UIUC Users without author permissio