CloQ, a prenyltransferase involved in clorobiocin biosynthesis

doi:10.1073/pnas.0337708100

CloQ, a prenyltransferase involved in clorobiocin biosynthesis

^*Pharmazeutische Biologie, Auf der Morgenstelle 8, and ^†Institut für Pharmakologie, Otfried-Müller-Strasse 10, Universität Tübingen, 72076 Tübingen, Germany; and ^‡Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Contributed by Christopher T. Walsh

Abstract

Ring A (3-dimethylallyl-4-hydroxybenzoic acid) is a structural moiety of the aminocoumarin antibiotics novobiocin and clorobiocin. In the present study, the prenyltransferase involved in the biosynthesis of this moiety was identified from the clorobiocin producer (Streptomyces roseochromogenes), overexpressed, and purified. It is a soluble, monomeric 35-kDa protein, encoded by the structural gene cloQ. 4-Hydroxyphenylpyruvate and dimethylallyl diphosphate were identified as the substrates of this enzyme, with K _m values determined as 25 and 35 μM, respectively. A gene inactivation experiment confirmed that cloQ is essential for ring A biosynthesis. Database searches did not reveal any similarity of CloQ to known prenyltransferases, and the enzyme did not contain the typical prenyl diphosphate binding site (N/D)DXXD. In contrast to most of the known prenyltransferases, the enzymatic activity was not dependent on the presence of magnesium, and in contrast to the membrane-bound polyprenyltransferases involved in ubiquinone biosynthesis, CloQ did not accept 4-hydroxybenzoic acid as substrate. CloQ and the similar NovQ from the novobiocin producer seem to belong to a new class of prenyltransferases.

Footnotes

↵ § To whom correspondence should be addressed. E-mail: heide{at}uni-tuebingen.de.
Data deposition: The sequences reported in this paper have been deposited in the GenBank database [accession nos. AF329398 (CloQ) and AAF67510 (NovQ)].
Abbreviations:

ring A,

3-dimethylallyl-4-hydroxybenzoic acid;

ring B of novobiocin,

3-amino-4,7-dihydroxy-8-methylcoumarin;

DMAPP,

dimethylallyl diphosphate;

4HPP,

4-hydroxyphenylpyruvate;

4HB,

4-hydroxybenzoic acid;

4HBAL,

4-hydroxybenzaldehyde;

3DMA,

3-dimethylallyl;

LC,

liquid chromatography;

ESI,

electrospray ionization;

CID,

collision-induced dissociation mass spectroscopy;

DMAT,

dimethylallyltryptophan synthase