SB-219383, a Novel Tyrosyl tRNA Synthetase Inhibitor from a Micromonospora sp. I. Fermentation, Isolation and Properties.

Stefanska, Anna L.; Coates, Nigel; Mensah, Lucy; Pope, Andrew J.; Ready, Sarah J.; Warr, S. R. C.

doi:10.7164/antibiotics.53.345

Cited by 49 publications

(24 citation statements)

References 8 publications

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“…These genes may have physiological roles in dealing with harsh environments or be directly involved in biosynthetic pathways given the huge metabolic potential of this group of bacteria. For example, several structurally complex natural products from actinomycetes are potent aaRS inhibitors, and functional genomic analyses of the corresponding strains can offer a means to explore the relationship between duplicated aaRSs and identify antibiotic biosynthetic gene clusters (12,29,30). The identification of two serS genes in the antibiotic albomycin producer Streptomyces sp.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

Zeng

Roy

Patil

et al. 2009

Antimicrob Agents Chemother

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The Trojan horse antibiotic albomycin, produced by Streptomyces sp. strain ATCC 700974, contains a thioribosyl nucleoside moiety linked to a hydroxamate siderophore through a serine residue. The seryl nucleoside structure (SB-217452) is a potent inhibitor of seryl-tRNA synthetase (SerRS) in the pathogenic bacterium Staphylococcus aureus, with a 50% inhibitory concentration (IC 50 ) of ϳ8 nM. In the albomycinproducing Streptomyces sp., a bacterial SerRS homolog (Alb10) was found to be encoded in a biosynthetic gene cluster in addition to another serRS gene (serS1) at a different genetic locus. Alb10, named SerRS2 herein, is significantly divergent from SerRS1, which shows high homology to the housekeeping SerRS found in other Streptomyces species. We genetically and biochemically characterized the two genes and the proteins encoded. Both genes were able to complement a temperature-sensitive serS mutant of Escherichia coli and allowed growth at a nonpermissive temperature. serS2 was shown to confer albomycin resistance, with specific amino acid residues in the motif 2 signature sequences of SerRS2 playing key roles. SerRS1 and SerRS2 are comparably efficient in vitro, but the K m of serine for SerRS2 measured during tRNA aminoacylation is more than 20-fold higher than that for SerRS1. SB-217452 was also enzymatically generated and purified by two-step chromatography. Its IC 50 against SerRS1 was estimated to be 10-fold lower than that against SerRS2. In contrast, both SerRSs displayed comparable inhibition kinetics for serine hydroxamate, indicating that SerRS2 was specifically resistant to SB-217452. These data suggest that mining Streptomyces genomes for duplicated aminoacyltRNA synthetase genes could provide a novel approach for the identification of natural products targeting aminoacyl-tRNA synthetases.

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Section: Discussionmentioning

confidence: 99%

Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

Zeng

Roy

Patil

et al. 2009

Antimicrob Agents Chemother

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“…The results presented in this study support this hypothesis and suggest that in order to design compounds that selectively inhibit bacterial tyrosyl-tRNA synthetases, one should take advantage of differences in the ATP-binding sites of the human and bacterial enzymes. Further support for this hypothesis comes from the observation that a species of Micromonospora produces a highly selective inhibitor of bacterial tyrosyl-tRNA synthetases, binding to Staphylococcus aureus tyrosyl-tRNA synthetases with a 40,000-fold higher affinity than it binds to the Saccharomyces cerevisiae homologue (9). The structure of this inhibitor consists of a tyrosyl moiety covalently attached to a bicyclic sugar through a peptide bond (5).…”

Section: Potassium Functionally Replaces the Second Lysine In The Kmsmentioning

confidence: 99%

“…Aminoacyl-tRNA synthetases have gained attention recently as potential targets for antibiotics (1)(2)(3)(4)(5)(6)(7)(8)(9)(10). Identifying differences in the catalytic mechanisms of bacterial and human aminoacyl-tRNA synthetases will facilitate the development of antibiotics that selectively target the bacterial aminoacyltRNA synthetases.…”

mentioning

confidence: 99%

Potassium Functionally Replaces the Second Lysine of the KMSKS Signature Sequence in Human Tyrosyl-tRNA Synthetase

Austin¹,

First

2002

Journal of Biological Chemistry

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Unlike their bacterial homologues, a number of eukaryotic tyrosyl-tRNA synthetases require potassium to catalyze the aminoacylation reaction. In addition, the second lysine in the class I-specific KMSKS signature motif is absent from all known eukaryotic tyrosyl-tRNA synthetase sequences, except those of higher plants. This lysine, which is the most highly conserved residue in the class I aminoacyl-tRNA synthetase family, has been shown to interact with the pyrophosphate moiety of the ATP substrate in the Bacillus stearothermophilus tyrosyl-tRNA synthetase. Equilibrium dialysis and presteady-state kinetic analyses were used to determine the role that potassium plays in the tyrosine activation reaction in the human tyrosyl-tRNA synthetase and whether it can be replaced by any of the other alkali metals. Kinetic analyses indicate that potassium interacts with the pyrophosphate moiety of ATP, stabilizing the E⅐Tyr⅐ATP and E⅐[Tyr-ATP] ‡ complexes by 2.3 and 4.3 kcal/mol, respectively. Potassium also appears to stabilize the asymmetric conformation of the human tyrosyltRNA synthetase dimer by 0.7 kcal/mol. Rubidium is the only other alkali metal that can replace potassium in catalyzing tyrosine activation, although the forward rate constant is half of that observed when potassium is present. The above results are consistent with the hypothesis that potassium functionally replaces the second lysine in the KMSKS signature sequence. Possible implications of these results with respect to the design of antibiotics that target bacterial aminoacyl-tRNA synthetases are discussed.Aminoacyl-tRNA synthetases have gained attention recently as potential targets for antibiotics (1-10). Identifying differences in the catalytic mechanisms of bacterial and human aminoacyl-tRNA synthetases will facilitate the development of antibiotics that selectively target the bacterial aminoacyltRNA synthetases. We are currently investigating the catalytic mechanisms of the human and Bacillus stearothermophilus tyrosyl-tRNA synthetases to elucidate the differences between these two enzymes.Tyrosyl-tRNA synthetase catalyzes the attachment of tyrosine to tyrosine tRNA (tRNA Tyr ) 1 by an ATP-dependent twostep reaction mechanism. In the first step, tyrosine is activated by MgATP to form an enzyme-bound tyrosyl-adenylate intermediate. The second step consists of the transfer of tyrosine to the 3Ј end of tRNA Tyr .TyrRS ϩ Tyr ϩ MgATP^TyrRS ⅐ Tyr-AMP ϩ PP i (Eq. 1)Tyrosyl-tRNA synthetase is a homodimer that displays "halfof-the-sites" reactivity, with only one tyrosyl-adenylate molecule formed per dimer (11-13). Although most of the active site amino acids are conserved between the human and B. stearothermophilus tyrosyl-tRNA synthetases, several differences exist between the two enzymes, including the inability of the human and bacterial tyrosyl-tRNA synthetases to aminoacylate each other's tRNA Tyr (14,15). In addition, sequence analyses indicate that the human tyrosyl-tRNA synthetase is Ͻ16% identical to the B. stearothermophilus enzyme and that four...

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“…The aminoacyl-tRNA synthetases have drawn interest as potential targets for antibiotics (32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45). In particular, Stefanska et al (37) have recently isolated a series of related competitive inhibitors of tyrosyl-tRNA synthetase that bind 40,000-fold more tightly to Staphyloccocus aureus tyrosyl-tRNA synthetase than it does to the S. cerevisiae enzyme (39 -41, 46 -50).…”

Section: Discussionmentioning

confidence: 99%

“…In particular, Stefanska et al (37) have recently isolated a series of related competitive inhibitors of tyrosyl-tRNA synthetase that bind 40,000-fold more tightly to Staphyloccocus aureus tyrosyl-tRNA synthetase than it does to the S. cerevisiae enzyme (39 -41, 46 -50). The selectivity of these inhibitors suggests that differences exist between the active sites of bacterial and eukaryotic tyrosyl-tRNA synthetases.…”

Section: Discussionmentioning

confidence: 99%

Catalysis of Tyrosyl-Adenylate Formation by the Human Tyrosyl-tRNA Synthetase

Austin

First

2002

Journal of Biological Chemistry

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Although the active site residues in the Bacillus stearothermophilus and human tyrosyl-tRNA synthetases are largely conserved, several differences exist between the two enzymes. In particular, three amino acids that stabilize the transition state for the activation of tyrosine in B. stearothermophilus tyrosyl-tRNA synthetase (Cys-35, His-48, and Lys-233) are not present in the human enzyme. This raises the question of whether the activation energy for the tyrosine activation step is higher for the human tyrosyl-tRNA synthetase than for the B. stearothermophilus enzyme. In this paper, we demonstrate that intrinsic fluorescence changes can be used to monitor the pre-steady state kinetics of human tyrosyl-tRNA synthetase. In contrast to the B. stearothermophilus enzyme, catalysis of the tyrosine activation step is potassium-dependent in the human tyrosyltRNA synthetase. Specifically, potassium increases the forward rate constant for tyrosine activation 260-fold in the human tyrosyl-tRNA synthetase. Comparison of the forward rate constants for catalysis of tyrosine activation by the human and B. stearothermophilus enzymes indicates that despite differences in their active sites and the potassium requirement of the human enzyme, the activation energies for tyrosine activation are identical for the two enzymes. The results of these investigations suggest that differences exist between the active sites of the bacterial and human tyrosyl-tRNA synthetases that could be exploited to design antimicrobials that target the bacterial enzyme.Aminoacyl-tRNA synthetases catalyze the transfer of amino acids to tRNA by a two-step reaction. In the first step, the amino acid substrate reacts with MgATP to form the enzymebound aminoacyl-adenylate intermediate. In the second step of the reaction, the aminoacyl moiety is transferred from the aminoacyl-adenylate intermediate to the 3Ј end of tRNA. There are two distinct classes of aminoacyl-tRNA synthetases. The Class I aminoacyl-tRNA synthetase family, of which tyrosyltRNA synthetase is a member, is characterized by an aminoterminal Rossmann-fold and conserved "HIGH" and "KMSKS" signature sequences. In tyrosyl-tRNA synthetase, the conserved HIGH and KMSKS signature sequences stabilize the transition state for the first step of the reaction (1-8).Bacillus stearothermophilus tyrosyl-tRNA synthetase is a homodimeric enzyme that displays "half-of-the-sites" reactivity with respect to tyrosine binding and tyrosyl-adenylate formation (9 -11). Site-directed mutagenesis and pre-steady state kinetic analyses have been used to identify 18 active site amino acids that stabilize the transition state for tyrosine activation in the B. stearothermophilus enzyme (reviewed in Refs. 12 and 13). Four of these amino acids are absent in the human tyrosyltRNA synthetase. In the B. stearothermophilus enzyme, replacement of three of these amino acids, Cys-35, His-48, and Lys-233, destabilizes the transition state for tyrosine activation by 1.2, 1.2, and 3.0 kcal/mol, respectively (1-6, 14). His-48 and Lys-233...

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SB-219383, a Novel Tyrosyl tRNA Synthetase Inhibitor from a Micromonospora sp. I. Fermentation, Isolation and Properties.

Cited by 49 publications

References 8 publications

Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

Characterization of Two Seryl-tRNA Synthetases in Albomycin-Producing Streptomyces sp. Strain ATCC 700974

Potassium Functionally Replaces the Second Lysine of the KMSKS Signature Sequence in Human Tyrosyl-tRNA Synthetase

Catalysis of Tyrosyl-Adenylate Formation by the Human Tyrosyl-tRNA Synthetase

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