The high‐resolution crystal structure of a 24‐kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Tsai, Francis T.F.; Singh, Onkar; Skarżyński, T.; Wonacott, A.; Weston, Simon C.; Tucker, Alec D.; Pauptit, Richard A.; Breeze, Alexander L.; Poyser, J. Philip; Ladbury, John E.; Wigley, Dale B.

doi:10.1002/(sici)1097-0134(199705)28:1<41::aid-prot4>3.3.co;2-b

Cited by 50 publications

(96 citation statements)

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“…The crystal structures of the bacterial 24-kDa N-terminal domain of GyrB (domain 1) in complex with numerous coumarins have been solved. However, this domain alone is monomeric and cannot bind ATP (17) The crystal structures of the full dimeric ATP binding domain (domain 1 and domain 2), also called the 43-kDa domain, in complex with ADPPNP and more recently with novobiocin (8,13,32,34,48,54) have been determined. Both structures show a dimeric organization of this enzyme.…”

Section: Resultsmentioning

confidence: 99%

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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Topoisomerase IV, a C 2 E 2 tetramer, is involved in the topological changes of DNA during replication. This enzyme is the target of antibacterial compounds, such as the coumarins, which target the ATP binding site in the ParE subunit, and the quinolones, which bind, outside the active site, to the quinolone resistance-determining region (QRDR). After site-directed and random mutagenesis, we found some mutations in the ATP binding site of ParE near the dimeric interface and outside the QRDR that conferred quinolone resistance to Streptococcus pneumoniae, a bacterial pathogen. Modeling of the N-terminal, 43-kDa ParE domain of S. pneumoniae revealed that the most frequent mutations affected conserved residues, among them His43 and His103, which are involved in the hydrogen bond network supporting ATP hydrolysis, and Met31, at the dimeric interface. All mutants showed a particular phenotype of resistance to fluoroquinolones and an increase in susceptibility to novobiocin. All mutations in ParE resulted in resistance only when associated with a mutation in the QRDR of the GyrA subunit. Our models of the closed and open conformations of the active site indicate that quinolones preferentially target topoisomerase IV of S. pneumoniae in its ATP-bound closed conformation.Topoisomerase IV and DNA gyrase are bacterial type II topoisomerases, which modify DNA topology during the replication process by unlinking DNA and facilitating chromosome segregation (59). Topoisomerase IV forms a C 2 E 2 tetramer involved in segregation of the chromosome at cell division (1, 30, 59). The ParC subunit contains the site of topoisomerization catalyzing the double-stranded DNA break (30, 53), while the ParE subunit catalyzes the hydrolysis of ATP, providing the free energy necessary for these reactions (2, 6, 11). The ParE and ParC subunits share extensive sequence homology with, respectively, the GyrB and GyrA subunits of the DNA gyrase A 2 B 2 tetramer, which catalyzes negative DNA supercoiling during the initiation and elongation processes of DNA replication (16,53).Both topoisomerases are the targets of antibacterial molecules, such as the quinolones and the coumarins. The coumarins inhibit supercoiling and enzyme turnover by preventing the binding and hydrolysis of ATP (45). The quinolones form a ternary complex with the topoisomerases in the presence of DNA, resulting in lethal double-stranded DNA breaks (11). Enzymatic studies and binding assays have also shown that the quinolones can form a complex with the topoisomerases before binding DNA (15, 29); however, some binding data indicate the occurrence of a specific and higher level of binding to the enzyme-DNA complex rather than to the enzyme alone (43,56). These families of molecules, in particular, the fluoroquinolones (FQs), are under continuous development as resistance to antibiotics in pathogenic bacteria has dramatically increased during the last decade (20,22,49).

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Section: Resultsmentioning

confidence: 99%

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

et al. 2003

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“…Simocyclinone possesses antimicrobial activity against Gram-positive bacteria, as well as exhibiting cytostatic effects against human tumor cell lines (6). However, its lack of a decorated noviosyl moiety at the 7-hydroxy position of the aminocoumarin scaffold suggests a molecular mechanism of action different than that of 1 and 2 given the X-ray information on GyrB complexes noted above for 1 and 2 (1,2). Indeed, recent studies reveal that simocyclinone D8 is a potent inhibitor of gyrase, albeit through a novel mode of action by preventing the initial binding of gyrase to DNA (Anthony Maxwell, personal communication, John Innes Centre, Norwich).…”

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Characterization of the Aminocoumarin Ligase SimL from the Simocyclinone Pathway and Tandem Incubation with NovM,P,N from the Novobiocin Pathway

et al. 2005

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Simocyclinone D 8 consists of an anguicycline C-glycoside tethered by a tetraene diester linker to an aminocoumarin. Unlike the antibiotics novobiocin, clorobiocin, and coumermycin A 1 , the phenolic hydroxyl group of the aminocoumarin in simocyclinone is not glycosylated with a decorated noviosyl moiety that is the pharmacophore for targeting bacterial DNA gyrase. We have expressed the Streptomyces antibioticus simocyclinone ligase SimL, purified it from Escherichia coli, and established its ATP-dependent amide bond forming activity with a variety of polyenoic acids including retinoic acid and fumagillin. We have then used the last three enzymes from the novobiocin pathway, NovM, NovP, and NovN, to convert a SimL product to a novel novobiocin analogue, in which the 3-prenyl-4-hydroxybenzoate of novobiocin is replaced with a tetraenoate moiety, to evaluate antibacterial activity.The aminocoumarin antibiotics novobiocin 1, clorobiocin 2, and coumermycin A 1 3, produced by various Streptomyces species (Figure 1), contain a bicyclic 3-amino-4,7-dihydroxycoumarin ring system, which serves as an essential scaffold for targeting them to the bacterial type II topoisomerases DNA gyrase and topoisomerase IV (1-5). Coumermycin A 1 is a pseudosymmetric dimer, containing elements of novobiocin (the 8-methylaminocoumarin) and clorobiocin (the 3′-O-methylpyrrolyl acyl group). Cocrystals of the N-terminal 24 kDa subfragment of the DNA gyrase B subunit (GyrB) 1 with 1 and 2 (1, 2) reveal that this family of antibiotics uses the aminocoumarin as a planar scaffold to present the decorated 4′-O-methyl-3′-O-acyl noviosyl moiety as the pharmacophore for inhibiting ATP hydrolysis in GyrB. These decorations of the L-deoxy sugar noviose include 4′-Omethylation and 3′-O-acylation with either a carbamoyl or methylpyrrolyl group. This 3′-O-acyl substituent is in intimate contact with GyrB active site residues and/or bound water molecules.Recently, another antibiotic containing the conserved aminocoumarin moiety, simocyclinone D 4 from Streptomyces antibioticus, has been discovered (6, 7). Simocyclinone D is a structurally unique antibiotic containing an aromatic anguicycline polyketide nucleus, the deoxy sugar D-olivose, and either the 8-chloro or 8-desmethyl version of the 3-amino-4,7-dihydroxycoumarin (8). The anguicycline Cglycoside and the aminocoumarin elements are linked by a tetraene dicarboxylic acid moiety. Simocyclinone possesses antimicrobial activity against Gram-positive bacteria, as well as exhibiting cytostatic effects against human tumor cell lines (6). However, its lack of a decorated noviosyl moiety at the 7-hydroxy position of the aminocoumarin scaffold suggests a molecular mechanism of action different than that of 1 and 2 given the X-ray information on GyrB complexes noted above for 1 and 2 (1, 2). Indeed, recent studies reveal that simocyclinone D8 is a potent inhibitor of gyrase, albeit through a novel mode of action by preventing the initial binding of gyrase to DNA (Anthony Maxwell, personal communicatio...

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“…X-ray crystallographic examination of antibiotic-enzyme complexes (15,18,39) showed that the ADHC and L-noviose moieties are each involved in antibiotic binding to the B subunit of DNA gyrase. The main difference between the structures of clorobiocin and novobiocin ( Fig.…”

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Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mutasynthesis

Galm¹,

Heller

Shapiro

et al. 2004

Antimicrob Agents Chemother

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Twenty-eight novel clorobiocin derivatives obtained from mutasynthesis experiments were investigated for their inhibitory activity towards Escherichia coli DNA gyrase and for their antibacterial activities towards clinically relevant gram-positive and gram-negative bacteria in comparison to novobiocin and clorobiocin. Clorobiocin was the most active compound both against E. coli DNA gyrase in vitro and against bacterial growth. All tested modifications of the 3-dimethylallyl-4-hydroxybenzoyl moiety reduced biological activity. The highest activities were shown by compounds containing a hydrophobic alkyl substituent at position 3 of the 4-hydroxybenzoyl moiety. Polar groups in this side chain, especially amide functions, strongly reduced antibacterial activity. Replacement of the alkyl side chain with a halogen atom or a methoxy group at the same position markedly reduced activity. Transfer of the pyrrole carboxylic acid moiety from O-3؆ to O-2؆ of L-noviose moderately reduced activity, whereas the complete absence of the pyrrole carboxylic acid moiety led to a loss of activity. Desclorobiocin derivatives lacking the chlorine atom at C-8 of the 3-amino-4,7-dihydroxycoumarin moiety also showed low activity. Lack of a methyl group at O-4؆ of L-noviose resulted in an inactive compound. From these findings it appears that clorobiocin represents a "highly evolved" structure optimized for bacterial transport and DNA gyrase inhibition.

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The high‐resolution crystal structure of a 24‐kDa gyrase B fragment from E. coli complexed with one of the most potent coumarin inhibitors, clorobiocin

Cited by 50 publications

References 22 publications

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

ATP-Bound Conformation of Topoisomerase IV: a Possible Target for Quinolones in Streptococcus pneumoniae

Characterization of the Aminocoumarin Ligase SimL from the Simocyclinone Pathway and Tandem Incubation with NovM,P,N from the Novobiocin Pathway

Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mutasynthesis

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