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

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