CloR, a Bifunctional Non-heme Iron Oxygenase Involved in Clorobiocin Biosynthesis

Abstract

The aminocoumarin antibiotics novobiocin and clorobiocin contain a 3-dimethylallyl-4-hydroxybenzoate (3DMA-4HB) moiety. The biosynthesis of this moiety has now been identified by biochemical and molecular biological studies. CloQ from the clorobiocin biosynthetic gene cluster in Streptomyces roseochromogenes DS 12976 has recently been identified as a 4-hydroxyphenylpyruvate-3-dimethylallyltransferase. In the present study, the enzyme CloR was overexpressed in Escherichia coli, purified, and identified as a bifunctional non-heme iron oxygenase, which converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) to 3DMA-4HB by two consecutive oxidative decarboxylation steps. In 18 O 2 labeling experiments we showed that two oxygen atoms are incorporated into the intermediate 3DMA-4HMA in the first reaction step, but only one further oxygen is incorporated into the final product 3DMA-4HB during the second reaction step. CloR does not show sequence similarity to known oxygenases. It apparently presents a novel member of the diverse family of the non-heme iron (II) and ␣-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an ␣-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature.

Activation of Dioxygen by Iron and Manganese Complexes: A Heme and Nonheme Perspective

“…133 The synthetic Fe II complex [Fe II (Tp Ph2 )(benzilate)] reacted with O 2 in benzene to form an Fe III -phenolate complex (λ max = 600 nm), along with the quantitative formation of benzophenone (generated from the decarboxylation of benzilate) (Scheme 12). 132 The proposed mechanism for this reaction involves initial formation of an Fe III (O 2 − ) species that abstracts a H-atom from the hydroxyl group to generate an Fe III OOH complex.…”

Go it alone: four-electron oxidations by mononuclear non-heme iron enzymes

“…The first decarboxylation converts 3DMA-HPP to 3-dimethylallyl-4-hydroxymandelate (3DMA-HMA) and the second produces 3DMA-HB (Fig. 1a) [36]. As 3DMAHMA is an isolable intermediate, the enzyme is likely to be distributive.…”

Enzymatic C–C-Coupling Prenylation: Bioinformatics – Modelling – Mechanism – Protein-Redesign – Biocatalytic Application

“…[25] The second group of aromatic prenyltransferases [26] consists of soluble enzymes with no sequence similarity to the enzymes of the first class. So far, four members have been biochemically characterised: CloQ, [27,28] involved in clorobiocin biosynthesis, NphB [29] (naphterpin biosynthesis), Fnq26 [30,31] (furanonaphthochinon I biosynthesis) and SCO7190. [29] None of these enzymes contains aspartate-rich motifs, though the activity of NphB depends on Mg 2+ .…”