Molecular characterization of the C-Methyltransferase NovO of Streptomyces spheroides, a valuable enzyme for performing Friedel-Crafts alkylation

摘要: Abstract The methyltransferase NovO cloned from Streptomyces spheroides could be heterologously produced as soluble and active enzyme in Escherichia coli . Sequencing of the novO gene revealed differences to GenBank entry AAF67508.1 resulting a different amino acid at position 223 (Cys instead Ser). A generated variant containing Ser residue this position, however, resulted poor ability express enzymatically protein. Characterization type I that performs its action dimer solution. Functional elements include conserved S-adenosyl- l -methionine (SAM) binding site (consensus: E/DXXXGXG) DLCCGSG (residues 45–51). Mutation analyses respective acids verified their importance for cofactor activity. In protein fractions mutants D45N G49A calculated k cat values decreased 2.5 × 10 −2  s −1 wild-type 9.7 × 10 −4 1.2 × 10 −3 , respectively. histidine 15 was identified catalytic base methyl transfer reaction. analysis purified preparations showed allyl groups via SAM analog allyl-SAH occurs with fourfold increased 11 × 10 compared 3.2 × 10 transfer. However, evolutionary design toward is obvious K m value 0.06 mM 0.22 mM allyl-SAH.