The aminocoumarin class of antibiotics, exemplified by novobiocin, is composed of tripartite
L-noviosylaminocoumarin prenylbenzoate natural products. The decorated noviosyl sugar componentinteracts with the target bacterial enzyme DNA gyrase. We have subcloned the putative 40 kDa
L-noviosyltransferase from
Streptomyces spheroides into
Escherichia coli, expressed it in soluble form, and purifiedit to homogeneity as a C-terminal His
8 fusion protein. The aglycone novobiocic acid, obtained from selectivedegradation 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 novobiocinintermediate with a
kcat of >300 min
-1. NovM displays activity with variant coumarin aglycones, suggestingit may be a promiscuous catalyst for noviosylation of a range of planar scaffolds. Conversely, NovMshows no activity with and is inhibited by TDP-
L-rhamnose (
Ki = 83.5 ± 5.5
M), the sugar donor thatmost closely structurally resembles the natural substrate TDP-
L-noviose. The NovM reaction productsgenerated during the course of this work will serve as substrates for subsequent analysis of the NovP andNovN tailoring enzymes that impart the noviose decorations required for DNA gyrase binding and antibioticactivity.