The Biosynthetic Gene Clusters of Aminocoumarin Antibiotics

Abstract

Plants and microorganisms are the most important sources of secondary metabolites in nature. For research in the functional genomics of secondary metabolism, and for the biotechnological application of such research by genetic engineering and combinatorial biosynthesis, most microorganisms offer a unique advantage to the researcher: the biosynthetic genes for a specific secondary metabolite are not scattered over the genome, but rather are clustered in a well-defined, contiguous region - the biosynthetic gene cluster of that metabolite. This is exemplified in this review for the biosynthetic gene clusters of the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A₁, which are potent inhibitors of DNA gyrase. Cloning, sequencing and analysis of the biosynthetic gene clusters of these three antibiotics revealed that the structural differences and similarities of the compounds are perfectly reflected by the genetic organisation of the biosynthetic gene clusters. The function of most biosynthetic genes could be identified by gene inactivation experiments as well as by heterologous expression and biochemical investigation. The prenylated benzoic acid moiety of novobiocin and clorobiocin, involved in the interaction with gyrase, is structurally similar to metabolites found in plants. However, detailed investigations of the biosynthesis revealed that the biosynthetic pathway and the enzymes involved are totally different from those identified in plants.

Antibiotics - aminocoumarins - biosynthesis - gene clusters - gyrase inhibitors - Streptomyces

References

• 1 Maxwell A, Lawson D M. The ATP-binding site of type II topoisomerases as a target for antibacterial drugs.  Curr Top Med Chem. 2003;  3 283-303
• 2 Steffensky M, Mühlenweg A, Wang Z -X, Li S -M, Heide L. Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11 891.  Antimicrob Agents Chemother. 2000;  44 1214-22
• 3 Wang Z -X, Li S -M, Heide L. Identification of the coumermycin A1 biosynthetic gene cluster of Streptomyces rishiriensis DSM 40 489.  Antimicrob Agents Chemother. 2000;  44 3040-8
• 4 Pojer F, Li S -M, Heide L. Molecular cloning and sequence analysis of the clorobiocin biosynthetic gene cluster: new insights into the biosynthesis of aminocoumarin antibiotics.  Microbiology. 2002;  148 3901-11
• 5 Bunton C A, Kenner G W, Robinson M JT, Webster B R. Experiments related to the biosynthesis of novobiocin and other coumarins.  Tetrahedron. 1963;  19 1001-10
• 6 Chen H, Walsh C T. Coumarin formation in novobiocin biosynthesis: β-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI.  Chem Biol. 2001;  8 301-12
• 7 Pacholec M, Hillson N J, Walsh C T. NovJ/NovK catalyze benzylic oxidation of a β-hydroxyl tyrosyl-S-pantetheinyl enzyme during aminocoumarin ring formation in novobiocin biosynthesis.  Biochemistry. 2005;  44 12 819-26
• 8 Holzenkämpfer M, Zeeck A. Biosynthesis of simocyclinone D8 in an ¹⁸O₂-rich atmosphere.  J Antibiot. 2002;  55 341-2
• 9 Eustáquio A S, Gust B, Luft T, Li S -M, Chater K F, Heide L. Clorobiocin biosynthesis in Streptomyces. Identification of the halogenase and generation of structural analogs.  Chem Biol. 2003;  10 279-88
• 10 Pacholec M, Tao J, Walsh C T. CouO and NovO: C-methyltransferases for tailoring the aminocoumarin scaffold in coumermycin and novobiocin antibiotic biosynthesis.  Biochemistry. 2005;  44 14 969-76
• 11 Mühlenweg A, Melzer M, Li S -M, Heide L. 4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase.  Planta. 1998;  205 407-13
• 12 Löscher R, Heide L. Biosynthesis of p-hydroxybenzoate from p-coumarate and p-coumaroyl-coenzyme A in cell-free extracts of Lithospermum erythrorhizon cell cultures.  Plant Physiol. 1994;  106 271-9
• 13 Melzer M, Heide L. Characterization of polyprenyldiphosphate: 4-hydroxybenzoate polyprenyltransferase from Escherichia coli .  Biochim Biophys Acta. 1994;  1212 93-102
• 14 Pojer F, Wemakor E, Kammerer B, Chen H, Walsh C T, Li S -M. et al . CloQ, a prenyltransferase involved in clorobiocin biosynthesis.  Proc Natl Acad Sci USA. 2003;  100 2316-21
• 15 Kuzuyama T, Noel J P, Richard S B. Structural basis for the promiscuous biosynthetic prenylation of aromatic natural products.  Nature. 2005;  435 983-7
• 16 Kawasaki T, Hayashi Y, Kuzuyama T, Furihata K, Itoh N, Seto H. et al . Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster.  J Bacteriol. 2006;  188 1236-44
• 17 Pojer F, Kahlich R, Kammerer B, Li S -M, Heide L. CloR, a bifunctional non-heme iron oxygenase involved in clorobiocin biosynthesis.  J Biol Chem. 2003;  278 30 661-8
• 18 Li S -M, Hennig S, Heide L. Biosynthesis of the dimethylallyl moiety of novobiocin via a non-mevalonate pathway.  Tetrahedron Lett. 1998;  39 2717-20
• 19 Xu H, Wang Z X, Schmidt J, Heide L, Li S -M. Genetic analysis of the biosynthesis of the pyrrole and carbamoyl moieties of coumermycin A1 and novobiocin.  Mol Genet Genomics. 2002;  268 387-96
• 20 Thomas M G, Burkart M D, Walsh C T. Conversion of L-proline to pyrrolyl-2-carboxyl-S-PCP during undecylprodigiosin and pyoluteorin biosynthesis.  Chem Biol. 2002;  9 171-84
• 21 Thuy T TT, Lee H C, Kim C G, Heide L, Sohng J K. Functional characterizations of novWUS involved in novobiocin biosynthesis from Streptomyces spheroides .  Arch Biochem Biophys. 2005;  436 161-7
• 22 Jakimowicz P, Tello M, Meyers C L, Walsh C T, Buttner M J, Field R A. et al . The 1.6-Å resolution crystal structure of NovW: a 4-keto-6-deoxy sugar epimerase from the novobiocin biosynthetic gene cluster of Streptomyces spheroides .  Proteins. 2006;  63 261-5
• 23 Freitag A, Li S -M, Heide L. Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU .  Microbiology. 2006;  152 2433-42
• 24 Steffensky M, Li S -M, Heide L. Cloning, overexpression, and purification of novobiocic acid synthetase from Streptomyces spheroides NCIMB 11 891.  J Biol Chem. 2000;  275 21 754-60
• 25 Galm U, Dessoy M A, Schmidt J, Wessjohann L A, Heide L. In vitro and in vivo production of new aminocoumarins by a combined biochemical, genetic and synthetic approach.  Chem Biol. 2004;  11 173-83
• 26 Schmutz E, Steffensky M, Schmidt J, Porzel A, Li S -M, Heide L. An unusual amide synthetase (CouL) from the coumermycin A1 biosynthetic gene cluster from Streptomyces rishiriensis DSM 40 489.  Eur J Biochem. 2003;  270 4413-9
• 27 Freel Meyers C L, Oberthur M, Anderson J W, Kahne D, Walsh C T. Initial characterization of novobiocic acid noviosyl transferase activity of NovM in biosynthesis of the antibiotic novobiocin.  Biochemistry. 2003;  42 4179-89
• 28 Albermann C, Soriano A, Jiang J, Vollmer H, Biggins J B, Barton W A. et al . Substrate specificity of NovM: Implications for novobiocin biosynthesis and glycorandomization.  Org Lett. 2003;  5 933-6
• 29 Freel Meyers C L, Oberthuer M, Xu H, Heide L, Kahne D, Walsh C T. Characterization of NovP and NovN: Completion of novobiocin biosynthesis by sequential tailoring of the noviosyl ring.  Angew Chem Int Ed Engl. 2004;  43 67-70
• 30 Xu H, Heide L, Li S -M. New aminocoumarin antibiotics formed by a combined mutational and chemoenzymatic approach utilizing the carbamoyltransferase NovN.  Chem Biol. 2004;  11 655-62
• 31 Freitag A, Wemakor E, Li S -M, Heide L. Acyl transfer in clorobiocin biosynthesis: Involvement of several proteins in the transfer of the pyrrole-2-carboxyl moiety to the deoxysugar.  Chembiochem. 2005;  6 2316-25
• 32 Freitag A, Rapp H, Heide L, Li S -M. Metabolic engineering of aminocoumarins: Inactivation of the methyltransferase gene cloP and generation of new clorobiocin derivatives in a heterologous host.  Chembiochem. 2005;  6 1411-8
• 33 Westrich L, Heide L, Li S -M. CloN6, a novel methyltransferase catalysing the methylation of the pyrrole-2-carboxyl moiety of clorobiocin.  Chembiochem. 2003;  4 768-73
• 34 Sofia H J, Chen G, Hetzler B G, Reyes-Spindola J F, Miller N E. Radical SAM, a novel protein superfamily linking unresolved steps in familiar biosynthetic pathways with radical mechanisms: functional characterization using new analysis and information visualization methods.  Nucleic Acids Res. 2001;  29 1097-106
• 35 Schmutz E, Mühlenweg A, Li S -M, Heide L. Resistance genes of aminocoumarin producers: Two type II topoisomerase genes confer resistance against coumermycin A1 and clorobiocin.  Antimicrob Agents Chemother. 2003;  47 869-77
• 36 Schmutz E, Hennig S, Li S -M, Heide L. Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParY^R and GyrB^R from the coumermycin A1 producer Streptomyces rishiriensis DSM 40 489.  Microbiology. 2004;  150 641-7
• 37 Eustáquio A S, Li S -M, Heide L. NovG, a DNA-binding protein acting as a positive regulator of novobiocin biosynthesis.  Microbiology. 2005;  151 1949-61
• 38 Peschke U, Schmidt H, Zhang H Z, Piepersberg W. Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78 - 11.  Mol Microbiol. 1995;  16 1137-56
• 39 Eustáquio A S, Luft T, Wang Z -X, Gust B, Chater K F, Li S -M. et al . Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation.  Arch Microbiol. 2003;  180 25-32
• 40 Eustáquio A S, Gust B, Galm U, Li S -M, Chater K F, Heide L. Heterologous expression of novobiocin and clorobiocin biosynthetic gene clusters.  Appl Environ Microbiol. 2005;  71 2452-9
• 41 Li S -M, Heide L. New aminocoumarin antibiotics from genetically engineered Streptomyces strains.  Curr Med Chem. 2005;  12 419-27