A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

Vecchione, James J.; Sello, Jason K.

doi:10.1128/aac.00723-09

Cited by 26 publications

(24 citation statements)

References 35 publications

(16 reference statements)

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“…All rotamers of an asparagine substitution at position 43 would result in the loss of two of the three hydrogen bonds observed in the network among indolmycin, His 43 , and Asp 132 . Similarly, the homologous H48Q mutation in Streptomyces coelicolor similarly appears incapable of forming both hydrogen bonds we observe for His 43 (36). Indolmycin can be modeled into the active site of H c TrpRS by rotating the oxazolinone ring away from Glu 199 to an orientation perpendicular to the indole moiety (Fig.…”

Section: Discussionmentioning

confidence: 99%

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Williams

Yin

Carter

2016

Journal of Biological Chemistry

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Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (K m ϳ 2 M), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ϳ1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg 2؉ ion in a manner that allows it to assist in transition state stabilization. The Mg 2؉ ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg 2؉ ⅐ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg 2؉ coordination in the human cytosolic (H c )TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in H c TrpRS with a yetto-be determined role for Mg 2؉ . Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to H c TrpRS.

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Section: Discussionmentioning

confidence: 99%

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Williams

Yin

Carter

2016

Journal of Biological Chemistry

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“…One gene codes for TrpRS, an enzyme sensitive to indolmycin (an antibiotic produced by S. griseus); the other gene, which is normally not expressed, codes for an enzyme insensitive to indolmycin (Vecchione & Sello, 2009). The presence of a family of paralogues in Streptomyces genomes has often been explained as allowing expression during different phases of the life cycle.…”

Section: Discussionmentioning

confidence: 99%

Acyl depsipeptide (ADEP) resistance in Streptomyces

2011

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ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEPinsensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.

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“…S. coelicolor strains were grown at 30°C on mannitol soya flour medium (SFM) or Difco nutrient agar medium (DNA) (24). For RNA isolation, S. avermitilis ATCC 31267 was grown in minimal liquid medium (NMMP) as previously described (40). Escherichia coli strains DH5␣ and ET12567/pUZ8002 were grown on Luria-Bertani medium at 37°C (36).…”

Section: Methodsmentioning

confidence: 99%

“…Since antibiotic resistance determinants accompany genes for antibiotic biosynthesis (7), it is a widely held notion that these bacteria represent a reservoir of antibiotic resistance genes (8,28,38). The mining of streptomycete genomes has led to the discovery of novel antibiotic resistance genes (40). The high frequency of antibiotic resistance observed in many Streptomyces species indicates that much lateral transfer of resistance genes has taken place (3,8,38,42).…”

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confidence: 99%

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Regulation of an Auxiliary, Antibiotic-Resistant Tryptophanyl-tRNA Synthetase Gene via Ribosome-Mediated Transcriptional Attenuation

Vecchione

Sello

2010

J Bacteriol

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cis-Acting RNA elements in the leaders of bacterial mRNA often regulate gene transcription, especially in the context of amino acid metabolism. We determined that the transcription of the auxiliary, antibioticresistant tryptophanyl-tRNA synthetase gene (trpRS1) in Streptomyces coelicolor is regulated by a ribosome-mediated attenuator in the 5 leader of its mRNA region. This regulatory element controls gene transcription in response to the physiological effects of indolmycin and chuangxinmycin, two antibiotics that inhibit bacterial tryptophanyl-tRNA synthetases. By mining streptomycete genome sequences, we found several orthologs of trpRS1 that share this regulatory element; we predict that they are regulated in a similar fashion. The validity of this prediction was established through the analysis of a trpRS1 ortholog (SAV4725) in Streptomyces avermitilis. We conclude that the trpRS1 locus is a widely distributed and self-regulating antibiotic resistance cassette. This study provides insights into how auxiliary aminoacyltRNA synthetase genes are regulated in bacteria.

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A Novel Tryptophanyl-tRNA Synthetase Gene Confers High-Level Resistance to Indolmycin

Cited by 26 publications

References 35 publications

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Acyl depsipeptide (ADEP) resistance in Streptomyces

Regulation of an Auxiliary, Antibiotic-Resistant Tryptophanyl-tRNA Synthetase Gene via Ribosome-Mediated Transcriptional Attenuation

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