The aminocoumarin class of antibiotics, exemplified by novobiocin, is composed of tripartite L-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar component interacts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa L-noviosyl transferase from Streptomyces spheroides into Escherichia coli, expressed it in soluble form, and purified it to homogeneity as a C-terminal His8 fusion protein. The aglycone novobiocic acid, obtained from selective degradation of novobiocin, and TDP-L-noviose, obtained by an 11-step chemical synthesis from L-rhamnose, were shown to be robust substrates for NovM to produce the desmethyldescarbamoyl novobiocin intermediate with a kcat of >300 min-1. NovM displays activity with variant coumarin aglycones, suggesting it may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovM shows no activity with and is inhibited by TDP-L-rhamnose (Ki = 83.5 ± 5.5 μM), the sugar donor that most closely structurally resembles the natural substrate TDP-L-noviose. The NovM reaction products generated during the course of this work will serve as substrates for subsequent analysis of the NovP and NovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibiotic activity.
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