CloQ, a prenyltransferase involved in clorobiocin biosynthesis

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 microM, 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.

Figure 1

Structures of the aminocoumarin antibiotics.

Figure 2

Possible biosynthetic pathways to the prenylated 4HB moiety (ring A) of novobiocin and clorobiocin.

Figure 3

HPLC analysis of culture extracts of the cloQ⁻ and cloI⁻ mutants. (A) Clorobiocin standard, wild type, cloQ⁻ mutant, and cloQ⁻ mutant fed with ring A. Detection was at 340 nm. The identity of clorobiocin ([M − H]⁻ = 695) was confirmed by LC-ESI-CID. Mass spectroscopic fragments are indicated. (B) Clorobiocin standard, wild type, and cloI⁻ mutant. Detection was at 340 nm. (C) Ring A standard, wild type, and cloI⁻ mutant. Detection was at 254 nm. The identity of ring A ([M − H]⁻ = 205) was confirmed by LC-ESI-CID. Mass spectroscopic fragments are indicated.

Figure 4

Purification of CloQ after overexpression as a fusion protein with GST. The SDS/12% polyacrylamide gel was stained with Coomassie brilliant blue. Lane 1, molecular mass standards; lane 2, total protein after isopropyl β-D-thiogalactoside induction; lane 3, soluble protein after induction; lane 4, eluate after thrombin treatment.

Figure 5

Radioactive prenyltransferase assays with CloQ. The three incubation mixtures were analyzed by HPLC with radioactivity detection. The retention times of tyrosine, β-OH-tyrosine, 4HBAL, 3DMA-4HBAL, and ring A were determined by using authentic reference samples.