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 18O2 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 α-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an α-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature. 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 18O2 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 α-ketoacid-dependent oxygenases, with 3DMA-4HPP functioning both as an α-keto acid and as a hydroxylation substrate. The reaction catalyzed by CloR represents a new pathway for the formation of benzoic acids in nature. The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 (Fig. 1) are natural products from Streptomyces spheroides NCIMB 11891 (syn. S. caeruleus) (1Lanoot B. Vancanneyt M. Cleenwerck I. Wang L. Li W. Liu Z. Swings J. Int. J. Syst. Evol. Microbiol. 2002; 52: 823-829PubMed Google Scholar), S. roseochromogenes DS 12.976, and S. rishiriensis DSM 40489, respectively (2Berger J. Batcho A.D. J. Chromatogr. Lib. 1978; 15: 101-159Crossref Scopus (35) Google Scholar). These antibiotics have been shown to target bacterial DNA gyrase, with binding constants (K d) in the range of 10 nm (3Maxwell A. Lawson D.M. Curr. Top. Med. Chem. 2003; 3: 283-303Crossref PubMed Scopus (230) Google Scholar, 4Kampranis S.C. Gormley N.A. Tranter R. Orphanides G. Maxwell A. Biochemistry. 1999; 38: 1967-1976Crossref PubMed Scopus (87) Google Scholar). Novobiocin (Albamycin®, Pfizer) is licensed for the treatment of infections with Gram-positive bacteria (5Montecalvo M.A. Horowitz H. Wormser G.P. Seiter K. Carbonaro C.A. Antimicrob. Agents Chemother. 1995; 39: 794Crossref PubMed Scopus (60) Google Scholar) and has been shown to enhance the cytotoxic activities of the anti-tumor drugs etoposide and teniposide (6Rappa G. Shyam K. Lorico A. Fodstad O. Sartorelli A.C. Oncol. Res. 2000; 12: 113-119Crossref PubMed Scopus (35) Google Scholar). So far, the therapeutic use of aminocoumarin antibiotics is limited due to their low solubility in water, toxicity in eukaryotes, and poor penetration in Gram-negative bacteria (7Maxwell A. Mol. Microbiol. 1993; 9: 681-686Crossref PubMed Scopus (229) Google Scholar). Combinatorial biosynthesis may offer a chance to develop novel aminocoumarins with improved properties (8Walsh C.T. Chembiochem. 2002; 3: 124-134Crossref Scopus (153) Google Scholar). Novobiocin and clorobiocin contain a prenylated 4-hydroxybenzoate moiety (called Ring A) (Fig. 1A). Recently, Lafitte et al. (9Lafitte D. Lamour V. Tsvetkov P.O. Makarov A.A. Klich M. Deprez P. Moras D. Briand C. Gilli R. Biochemistry. 2002; 41: 7217-7223Crossref PubMed Scopus (209) Google Scholar) demonstrated that Ring A plays a role in the binding affinity of these antibiotics for gyrase. Knowledge of the biosynthetic pathway of Ring A may facilitate future efforts to create structural modifications of this moiety by combinatorial biosynthesis. The dimethylallyl moiety of Ring A is derived from the methyl erythritol-4-phosphate (MEP) pathway (10Li S.-M. Hennig S. Heide L. Tetrahedron Lett. 1998; 39: 2717-2720Crossref Scopus (32) Google Scholar). The 4-hydroxybenzoic acid (4HB) 1The abbreviations used are: 4HB, 4-hydroxybenzoic acid; 4HPP, 4-hydroxyphenylpyruvate; 3DMA-4HBAL, 3-dimethylallyl-4-hydroxybenzaldehyde; 3DMA-4HMA, 3-dimethylallyl-4-hydroxymandelic acid; 3DMA-4HPP, 3-dimethylallyl-4-hydroxyphenylpyruvate; IPTG, isopropyl-1-thio-β-d-galactopyranoside; GST, glutathione S-transferase; LC, liquid chromatography; MS, mass spectrometry; MS-MS, tandem mass spectrometry; HPLC, high performance liquid chromatography; HmaS, 4-hydroxymandelate synthase; DMAPP, dimethylallyl diphosphate.1The abbreviations used are: 4HB, 4-hydroxybenzoic acid; 4HPP, 4-hydroxyphenylpyruvate; 3DMA-4HBAL, 3-dimethylallyl-4-hydroxybenzaldehyde; 3DMA-4HMA, 3-dimethylallyl-4-hydroxymandelic acid; 3DMA-4HPP, 3-dimethylallyl-4-hydroxyphenylpyruvate; IPTG, isopropyl-1-thio-β-d-galactopyranoside; GST, glutathione S-transferase; LC, liquid chromatography; MS, mass spectrometry; MS-MS, tandem mass spectrometry; HPLC, high performance liquid chromatography; HmaS, 4-hydroxymandelate synthase; DMAPP, dimethylallyl diphosphate. moiety can be formed in nature by at least three well-established mechanisms: (a) the direct conversion of chorismic acid to 4HB by chorismate pyruvate-lyase (11Siebert M. Severin K. Heide L. Microbiology. 1994; 140: 897-904Crossref PubMed Scopus (86) Google Scholar); (b) the removal of acetyl-CoA from the side chain of 4-coumaroyl-CoA by an oxidative reaction mechanism, analogous to the β-oxidation of fatty acids, resulting in 4-hydroxybenzoyl-CoA (12Löscher R. Heide L. Plant Physiol. 1994; 106: 271-279Crossref PubMed Scopus (83) Google Scholar, 13Hertweck C. Moore B.S. Tetrahedron. 2000; 56: 9115-9120Crossref Scopus (53) Google Scholar); and (c) the removal of a C2 unit from the side chain of a β-hydroxy-β-phenylpropionate derivative via a retro-aldol reaction; the resulting benzaldehyde derivate can subsequently be oxidized to the corresponding acid (14Gasson M.J. Kitamura Y. McLauchlan W.R. Narbad A. Parr A.J. Parsons E.L. Payne J. Rhodes M.J. Walton N.J. J. Biol. Chem. 1998; 273: 4163-4170Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 15Ahmed M.A. El-Mawla A. Beerhues L. Planta. 2002; 214: 727-733Crossref PubMed Scopus (80) Google Scholar). In novobiocin biosynthesis, feeding experiments with isotope-labeled precursors showed that 4-hydroxyphenylpyruvate and tyrosine are efficiently incorporated into Ring A (10Li S.-M. Hennig S. Heide L. Tetrahedron Lett. 1998; 39: 2717-2720Crossref Scopus (32) Google Scholar, 16Kominek L.A. Sebek O.K. Dev. Ind. Microbiol. 1974; 15: 60-69Google Scholar), ruling out the chorismate pyruvate-lyase reaction as the principal source of this compound. The aminocoumarin moiety of novobiocin (Ring B) is formed from tyrosine via the intermediate β-hydroxytyrosyl-S-NovH. Chen and Walsh (17Chen H. Walsh C.T. Chem. Biol. 2001; 8: 301-312Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) suggested that Ring A may also be formed from this intermediate, following the reaction mechanism shown in Fig. 2A, i.e. by a retroaldol reaction to 4-hydroxybenzaldehyde, oxidation to the acid and prenylation. 3-Prenylation of 4HB has a well-established precedent in ubiquinone biosynthesis (18Melzer M. Heide L. Biochim. Biophys. Acta. 1994; 1212: 93-102Crossref PubMed Scopus (99) Google Scholar). Cloning and sequencing of the biosynthetic gene clusters of novobiocin, clorobiocin, and coumermycin A1 (Fig. 1B) (19Pojer F. Li S.-M. Heide L. Microbiology. 2002; 148: 3901-3911Crossref PubMed Scopus (133) Google Scholar) first appeared to support this hypothesis. Three genes were identified in the novobiocin and clorobiocin clusters for which no homologues existed in the coumermycin cluster. We speculated that these genes might be involved in the biosynthesis of Ring A (which is absent in coumermycin A1). These genes were: (a) cloR and novR, which showed sequence similarity to putative aldolases; CloR was indeed shown to be involved in Ring A biosynthesis by a gene inactivation experiment (19Pojer F. Li S.-M. Heide L. Microbiology. 2002; 148: 3901-3911Crossref PubMed Scopus (133) Google Scholar); (b) cloF and novF, which show sequence similarities to dehydrogenases; and (c) cloQ and novQ, which did not show sequence similarities to known genes in the data base. We recently confirmed that CloQ codes for a prenyltransferase (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). The substrate of this enzyme, however, was none of the intermediates discussed above but, rather, 4-hydroxyphenylpyruvate (4HPP, Fig. 2B). The product of the reaction, i.e. 3-dimethylallyl-4HPP, was easily degraded to 3-dimethylallyl-4-hydroxybenzaldehyde (3DMA-4HBAL), suggesting a biosynthesis of Ring A by the mechanism depicted in Fig. 2B, i.e. via oxidation of 3DMA-4HBAL to the corresponding acid. In the present study, we investigated the conversion of 3DMA-4HPP to Ring A and the role of CloR in this reaction sequence. Unexpectedly, the results showed that the biosynthesis of Ring A follows neither of the two mechanisms depicted in Fig. 2. Rather, the conversion of 3DMA-4HPP to Ring A proceeds in two oxidative decarboxylation steps, via 3-dimethylallyl-4-hydroxymandelic acid (3DMA-4HMA) as intermediate, and is catalyzed by the bifunctional non-heme iron oxygenase CloR. This represents a new pathway for the formation of benzoic acids in nature. General Chemicals—4-Hydroxyphenylpyruvate (4HPP), d- and l-mandelic acid, dl-4-hydroxymandelic acid, and 4-hydroxyphenyllactic acid were purchased from Sigma. β-(R,S)-Hydroxy-l-tyrosine and DMAPP were kindly provided by Dr. K.-H. van Pée (Dresden, Germany) and Dr. K. Yazaki (Kyoto, Japan), respectively. 3-Dimethylallyl-4-hydroxybenzaldehyde (3DMA-4HBAL) was synthesized as described by Glüsenkamp and Büchi (21Gluesenkamp K.H. Buechi G. J. Org. Chem. 1986; 51: 4481-4483Crossref Scopus (20) Google Scholar). Ring A (3-dimethylallyl-4-hydroxybenzoic acid) was prepared as described previously (22Kominek L.A. Meyer H.F. Methods Enzymol. 1975; 43: 502-508Crossref PubMed Scopus (12) Google Scholar). Oxygen (18O2; 99%) was purchased from Campro Scientific (Berlin, Germany). DNA Manipulation—DNA manipulations and standard genetic techniques in Escherichia coli were carried out as described by Sambrook and Russell (23Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York2001Google Scholar). DNA fragments were isolated from agarose gels using a NucleoSpin 2 in 1 extraction kit (Macherey-Nagel, Düren, Germany). Isolation of plasmids was carried out with ion-exchange columns (Nucleobond AX kit, Macherey-Nagel). Cloning of cloR—For construction of the plasmid cloR-pGEX4T1, cloR was amplified using the primers cloR-Nterm-BamHI (5′-AGGTGCCCGGGATCCAAGGCTTTGGCGAAC-3′) and cloR-Cterm-XhoI (5′-TCGGCACTCGAGCCTTGCCAGGGCGGGCGTTTG-3′). Introduced restriction sites are underlined. The product was digested with BamHI and XhoI, gel purified and ligated into the same site of pGEX-4T-1 (Amersham Biosciences) to give cloR-pGEX4T1. The plasmid was transformed into E. coli strain BL21(DE3) for overexpression of CloR as GST (glutathione S-transferase)-tagged protein. Overexpression and Purification of CloR Protein—E. coli BL21(DE3)/cloR-pGEX4T1 cells were grown in of at an A of was was to a final of at the cells were by at and in 10 1 cells were by was by at Purification of CloR as a GST and of the GST by treatment were carried out to the (Amersham Biosciences) using and the Overexpression and Purification of of overexpression in E. coli and of CloQ were carried out as described in et al. (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). techniques were used as described in PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The molecular mass of CloR was by gel a that has been with The was with and A (Amersham of with CloQ the substrate for CloR reaction, was by CloQ with and DMAPP as described in et al. (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). A reaction 4HPP, DMAPP, and purified was at for 1 This reaction of was by of with 1 for at for the of with 3DMA-4HPP were as described for Walsh C.T. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Chem. 2000; Scopus Google Scholar) with The reaction of the CloQ reaction of 3DMA-4HPP, acid, and of for 1 at the reaction was with of acid. formation was by high performance liquid at nm for acid) with a at a of 1 A of in acid was A was carried out with of with of the substrate acid, 4-hydroxyphenyllactic acid) were used of 3DMA-4HPP, and was carried out by at nm with a of in acid at a of 1 were carried out with CloQ was also with acid 4-hydroxybenzoic acid and DMAPP as described in et al. (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). Isolation of of CloQ reaction product of acid, and of and was The reaction was with of acid and with 1 of the was in of 3DMA-4HMA was isolated by as described and was confirmed by mass in the of 2 one 3DMA-4HPP and one reaction were by of a and with for three The was to the substrate The was by of a and 18O2 was to into the for 1 at the reaction was with acid and by liquid to an mass in A of in acid was In a 18O2 labeling 3DMA-4HMA as described was used as substrate of of CloR and gene cloR from the clorobiocin cluster and the corresponding gene from the novobiocin cluster (Fig. 1B) of and acids, and show and A data sequence similarity to which are by J. Mol. Biol. PubMed Scopus Google Scholar) and M. D. Biol. 2002; PubMed Scopus (20) Google Scholar), and to the Y. J. S.C. Biochemistry. 2001; PubMed Scopus (35) Google Scholar). These contain a in their and are involved in the of in E. cloR is to the gene cloQ which the prenyltransferase involved in Ring A formation (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). The same is in the novobiocin cluster for the corresponding genes and and Purification of to CloR as a glutathione as an CloR showed sequence similarity to and these for J. Mol. Biol. 1993; PubMed Scopus Google Scholar). A using the can to a of the due to the and the E. coli cells CloR only grown at of cells were at and with GST was from CloR by treatment and This in apparently CloR as by (Fig. The molecular mass in to the mass of the A of 1 of CloR of was using gel the molecular mass of CloR was as that the was in of the of the CloR the of we first 3DMA-4HPP (Fig. the putative substrate of by of 4-hydroxyphenylpyruvate with DMAPP and the prenyltransferase CloR and and Microbiol. Lett. 2001; PubMed Google Scholar), were to the reaction no formation of (3DMA-4HBAL) Ring A be by A using a with and as however, the of a of a new product with a of in (Ring 3DMA-4HBAL, and 3DMA-4HPP, not This product was absent CloR was DMAPP were from the This that the new was derived from In the biosynthesis of in is to acid of the non-heme iron Walsh C.T. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Chem. 2000; Scopus Google Scholar). described by et al. Walsh C.T. Chem. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and to be by with an of and by has to be to by acid to an CloR did not show sequence similarity to non-heme we to CloR of CloR for with 1 acid and 3DMA-4HPP were for 1 the products of the reaction were by (Fig. these formation of the new product was in the of and an product was that showed the same as an Ring A in with an confirmed that this product was indeed Ring A product was isolated by and with in the of acid, the formation of Ring A was demonstrated by not that product was an intermediate in the formation of Ring A. of product that this showed the mass of 3-dimethylallyl-4-hydroxymandelic acid showed the of a acid derivative and the same was from an acid and CloR two consecutive (Fig. the conversion of 3DMA-4HPP to acid and the conversion of 3DMA-4HMA to acid, i.e. Ring A. CloR was to be for 3DMA-4HPP and 3DMA-4HMA as product formation was with the 4-hydroxyphenylpyruvate dl-4-hydroxymandelic acid, with acid, l-mandelic acid, 4-hydroxyphenyllactic acid. experiments confirmed that the prenyltransferase CloQ (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar) prenylated and was not to with dl-4-hydroxymandelic acid 4-hydroxybenzoic acid not of with in of of as described previously for non-heme iron L. Chem. PubMed Scopus Google Scholar). non-heme an as acid J. Chem. 2000; Scopus Google Scholar). the of CloR was not by of CloR was a protein. showed an at nm but no in the CloR is not a as the of the of of CloR for and suggested that to the non-heme iron L. Chem. PubMed Scopus Google Scholar, L. A. D. P. A. E. C. 1999; Full Text Full Text PDF Scopus Google Scholar, 2000; PubMed Scopus Google Scholar). indeed molecular oxygen was the substrate of the CloR reaction, and one both oxygen atoms of were incorporated into the we carried out experiments with of the was by In the first CloR with was with 3DMA-4HPP and in an 18O2 A was carried out in the The reaction products 3DMA-4HMA and 3DMA-4HB were by and by Fig. the molecular in a and in 18O2 3DMA-4HMA showed the molecular at In of the 3DMA-4HMA the 18O2 showed the molecular at the of two The of the was by the decarboxylation product was the that one of the atoms been incorporated into the of the direct decarboxylation product and the corresponding from oxidation were 2 the corresponding of the 3DMA-4HMA, that the 18O2 been incorporated into the the molecular of 3DMA-4HMA, also a at was (Fig. resulting from the of a into the This a of the and has been previously for as as for non-heme iron J. Chem. 2000; Scopus Google Scholar). This has been suggested to from an of a intermediate with J. Chem. 2000; Scopus Google Scholar). The product also 3DMA-4HMA due to the of in the In to 3DMA-4HMA, the CloR reaction also of two atoms into this product was demonstrated by the molecular at in to for the (Fig. both these were in the (Fig. a second labeling the intermediate 3DMA-4HMA was first in and isolated by This was in an 18O2 with CloR with and The resulting 3DMA-4HB was by (Fig. and this showed the of one into the of the as demonstrated by the molecular at and the decarboxylation product at (Fig. Fig. the results of the 18O2 atoms are incorporated in the first reaction step, and one is incorporated in the second reaction step. The involved in the second is to In the present study, CloR was identified as a bifunctional oxygenase that converts 3-dimethylallyl-4-hydroxyphenylpyruvate (3DMA-4HPP) in two consecutive reaction to 3-dimethylallyl-4-hydroxybenzoate i.e. to the Ring A moiety of 18O2 labeling experiment confirmed that molecular oxygen is used as substrate by CloR. The purified CloR does not contain a and by and that to the non-heme iron The first reaction catalyzed by i.e. the conversion of 3DMA-4HPP to 3-dimethylallyl-4-hydroxymandelic acid has a well-established precedent in the reaction in biosynthesis. to the and α-ketoacid-dependent in L. Chem. PubMed Scopus Google Scholar, 2000; PubMed Scopus Google Scholar, L. Biol. 2000; PubMed Scopus Google Scholar). These and an as the reaction, the and the is oxidized to a by of one of the oxygen atoms of The oxygen may be used for a hydroxylation reaction, by the and α-ketoacid-dependent have been shown to not only but a range of diverse oxidative and of these are bifunctional acid consecutive oxidative a biosynthetic In the conversion of 4-hydroxyphenylpyruvate to by as a substrate for hydroxylation and at the same as CloR first reaction and out a reaction as but the of the substrate of the sequence similarity to CloR does The second reaction catalyzed by CloR is the conversion of 3DMA-4HMA to It is to that this reaction may hydroxylation of the of 3DMA-4HMA, resulting in a which to give the corresponding i.e. decarboxylation of this in the final the reaction mechanism be to that of the first reaction and that of and α-ketoacid-dependent did not the of in the and the mechanism of the CloR reaction at It is that the catalyzed by and α-ketoacid-dependent a 2000; PubMed Scopus Google Scholar, In A. E. W. L. 2003; PubMed Scopus Google Scholar). this is for CloR to be The of and α-ketoacid-dependent sequence similarity to 2000; PubMed Scopus Google Scholar, L. Biol. 2000; PubMed Scopus Google Scholar), and is not that CloR does not show sequence similarity to known of this a data that CloR does show similarity to of from of These may of as CloR. The structural of and α-ketoacid-dependent is the L. Biol. 2000; PubMed Scopus Google Scholar, E.L. L. J. PubMed Scopus Google Scholar). It of two and one which the iron in the site of the of the sequence of CloR with that of as as with the of of the data of with high sequence similarity to CloR not and of CloR as These acids may be for a of this The of CloR in the biosynthesis of the prenylated 4-hydroxybenzoate moiety of clorobiocin was in by a gene inactivation experiment (19Pojer F. Li S.-M. Heide L. Microbiology. 2002; 148: 3901-3911Crossref PubMed Scopus (133) Google Scholar). The present results now the of a for the formation of this moiety of clorobiocin, as shown in Fig. CloQ is a which converts to 3DMA-4HPP (20Pojer F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google Scholar). CloR converts the CloQ reaction product to Ring as demonstrated in the present sequence similarity to and is to as the substrate for to of the biosynthetic gene cluster N.J. M. Chem. Biol. 1998; Full Text PDF PubMed Scopus Google Scholar). the coumermycin cluster does not contain a (Fig. feeding experiments with the novobiocin Spring J. PubMed Scopus Google Scholar) showed that was incorporated into Ring was incorporated in Ring A. This may that a tyrosine for the formation of Ring for Ring A biosynthesis, and that both via a reaction (Fig. Chen and Walsh (17Chen H. Walsh C.T. Chem. Biol. 2001; 8: 301-312Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) have previously speculated that Ring A and Ring of novobiocin may both be via as a CloQ and CloR F. Li S.-M. Heide L. Microbiology. 2002; 148: 3901-3911Crossref PubMed Scopus (133) Google and F. Wemakor E. Kammerer B. Chen H. Walsh C.T. Li S.-M. Heide L. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2316-2321Crossref PubMed Scopus (116) Google and the present however, now that these two of clorobiocin of novobiocin as are by two (Fig. The of CloR a new member to the diverse family of the iron and the of a new pathway for the formation of benzoic acids in nature. We Walsh for Wemakor for and for the of