Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin

Caren L Meyers, Markus Oberthür, John W. Anderson, Daniel Kahne, Christopher T. Walsh

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)4179-4189
Number of pages11
JournalBiochemistry®
Volume42
Issue number14
DOIs
StatePublished - Apr 15 2003
Externally publishedYes

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Novobiocin
Biosynthesis
Transferases
DNA Gyrase
Rhamnose
Anti-Bacterial Agents
Sugars
Acids
Aminocoumarins
Substrates
Bacterial DNA
Streptomyces
Enzymes
Biological Products
Reaction products
Scaffolds
Escherichia coli
Fusion reactions
Degradation
Catalysts

ASJC Scopus subject areas

  • Biochemistry

Cite this

Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin. / Meyers, Caren L; Oberthür, Markus; Anderson, John W.; Kahne, Daniel; Walsh, Christopher T.

In: Biochemistry®, Vol. 42, No. 14, 15.04.2003, p. 4179-4189.

Research output: Contribution to journalArticle

Meyers, Caren L ; Oberthür, Markus ; Anderson, John W. ; Kahne, Daniel ; Walsh, Christopher T. / Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin. In: Biochemistry®. 2003 ; Vol. 42, No. 14. pp. 4179-4189.
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