M.(phi)BssHII, a novel cytosine-C5-DNA-methyltransferase with target-recognizing domains at separated locations of the enzyme

In all cytosine-C5-DNA-methyltransferases (MTases) from prokaryotes and euk aryotes, remarkably conserved amino acid sequence elements responsible for general enzymatic functions are arranged in the same canonical order, In ad dition, one variable region, which includes the target-recognizing domain(s ) (TRDs) characteristic for each enzyme, has been localized in one region b etween the same blocks of these conserved elements. This conservation in th e order of conserved and variable sequences suggests stringent structural c onstraints in the primary structure to obtain the correct folding of the en zymes. Here we report the characterization of a new type of a multispecific MTase, M.(phi)BssHII, which is expressed as two isoforms, Isoform I is an entirely novel type of MTase which has, in addition to the TRDs at the conv entional location, one TRD located at a noncanonical position at its N-term inus. Isoform II is represented by the same MTase, but without the N-termin al TRD, The N-terminal TRD provides HaeII methylation specificity to isofor m I. The TRD is fully functional when engineered into either the convention al variable region of M.(phi)BssHII or the related monospecific M.phi 3TII MTase, The implications of this structural plasticity with respect to the e volution of MTases are discussed,