Identifying the targets of aminoacyl-tRNA synthetase inhibitors by primer extension inhibition

Aminoacyl-transfer RNA (tRNA) synthetases (RS) are essential components of the cellular translation machinery and can be exploited for antibiotic discovery. Because cells have many different RS, usually one for each amino acid, identification of the specific enzyme targeted by a new natural or synthetic inhibitor can be cumbersome. We describe the use of the primer extension technique in conjunction with specifically designed synthetic genes to identify the RS targeted by an inhibitor. Suppression of a synthetase activity reduces the amount of the cognate aminoacyl-tRNA in a cell-free translation system resulting in arrest of translation when the corresponding codon enters the decoding center of the ribosome. The utility of the technique is demonstrated by identifying a switch in target specificity of some synthetic inhibitors of threonyl-tRNA synthetase.     

Methionine analogues as inhibitors of methionyl-tRNA synthetase

A series of methionine analogues have been synthesized as inhibitors of methionyl-tRNA synthetase and evaluated for their inhibitory activities of E. coli methionyl-tRNA synthetase and bacterial growth. Among them, -methionine hydroxamate 20 has proved to be the best inhibitor of the enzyme with K_i = 19 μM and showed a growth inhibition against E.coli JM 109, P. vulganis 6059 and C. freundii 8090.     

Adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate are synthesized by yeast acetyl coenzyme A synthetase.

Yeast (Saccharomyces cerevisiae) acetyl coenzyme A (CoA) synthetase (EC 6.2.1.1) catalyzes the synthesis of adenosine 5'-tetraphosphate (P4A) and adenosine 5'-pentaphosphate (p5A) from ATP and tri- or tetrapolyphosphate (P3 or P4), with relative velocities of 7:1, respectively. Of 12 nucleotides tested as potential donors of nucleotidyl moiety, only ATP, adenosine-5'-O-[3-thiotriphosphate], and acetyl-AMP were substrates, with relative velocities of 100, 62, and 80, respectively. The Km values for ATP, P3, and acetyl-AMP were 0.16, 4.7, and 1.8 mM, respectively. The synthesis of p4A could proceed in the absence of exogenous acetate but was stimulated twofold by acetate, with an apparent Km value of 0.065 mM. CoA did not participate in the synthesis of p4A (p5A) and inhibited the reaction (50% inhibitory concentration of 0.015 mM). At pH 6.3, which was optimum for formation of p4A (p5A), the rate of acetyl-CoA synthesis (1.84 mumol mg-1 min-1) was 245 times faster than the rate of synthesis of p4A measured in the presence of acetate. The known formation of p4A (p5A) in yeast sporulation and the role of acetate may therefore be related to acetyl-CoA synthetase.     

Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase

Diadenosine-5',5'-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells. It produces 2.4 mg of homogeneous enzyme with a yield of 3.1%. The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 [37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2]. The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography. Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not). Among the products, a nucleoside diphosphate is always formed. ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is. Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold). With 50 microM MnCl2, the stimulation is 900-fold. Ca2+, Fe2+, and Mg2+ have no effect. The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+. The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP. However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+. Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of a GatCAB amidotransferase: aspartyl-tRNA synthetase increases its affinity for Asp-tRNA(Asn) and novel aminoacyl-tRNA analogues are competitive inhibitors

The trimeric GatCAB aminoacyl-tRNA amidotransferases catalyze the amidation of Asp-tRNAAsn and/or Glu-tRNAGln to Asn-tRNAAsn and/or Gln-tRNAGln, respectively, in bacteria and archaea lacking an asparaginyl-tRNA synthetase and/or a glutaminyl-tRNA synthetase. The two misacylated tRNA substrates of these amidotransferases are formed by the action of nondiscriminating aspartyl-tRNA synthetases and glutamyl-tRNA synthetases. We report here that the presence of a physiological concentration of a nondiscriminating aspartyl-tRNA synthetase in the transamidation assay decreases the Km of GatCAB for Asp-tRNAAsn. These conditions, which were practical for the testing of potential inhibitors of GatCAB, also allowed us to discover and characterize two novel inhibitors, aspartycin and glutamycin. These analogues of the 3'-ends of Asp-tRNA and Glu-tRNA, respectively, are competitive inhibitors of the transamidase activity of Helicobacter pylori GatCAB with respect to Asp-tRNAAsn, with Ki values of 134 microM and 105 microM, respectively. Although the 3' end of aspartycin is similar to the 3' end of Asp-tRNAAsn, this analogue was neither phosphorylated nor transamidated by GatCAB. These novel inhibitors could be used as lead compounds for designing new types of antibiotics targeting GatCABs, since the indirect pathway for Asn-tRNAAsn or Gln-tRNAGln synthesis catalyzed by these enzymes is not present in eukaryotes and is essential for the survival of the above-mentioned bacteria.     

Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.     

Methionyl adenylate analogues as inhibitors of methionyl-tRNA synthetase.

Four stable analogues of methionyl adenylate (3-6) were designed as inhibitors of methionyl-tRNA synthetase and synthesized from 2',3'-isopropylideneadenosine. They strongly inhibited aminoacylation activity of methionyl-tRNA synthetases isolated from Escherichia coli, Mycobacterium tuberculosis, Saccharomyces cerevisiae and human. Among the microorganisms tested, however, these chemicals showed the growth inhibition effect only on E. coli.     

Effect of diadenosine oligophosphates (Ap4A and Ap3A) and their phosphonate analogs on catalytic properties of phenylalanyl-tRNA synthetase from E. coli

The influence of P1,P3-bis(5'-adenosyl)triphosphate (Ap3A), P1,P4-bis(5'-adenosyl)tetraphosphate (Ap4A) and its analogues, containing a residue of methylenediphosphonic acid in various positions of the oligophosphate chain, on the reactions catalysed by phenylalanyl-tRNA synthetase from E. coli MRE-600 has been studied. The compounds do not affect significantly the rate of ATP-[32P]PPi-exchange nor maintain this reaction in the absence of ATP. The diadenosineoligophosphates are shown to be noncompetitive inhibitors of ATP in the tRNA aminoacylation by phenylalanine (for Ap4A Ki = 1,45.10(-3) M). The phosphonate analogues of Ap4A inhibit the synthesis of Ap3A depending on their structure. The conclusion is thus drawn that the E. coli MRE-600 phenylalanyl-tRNA synthetase does not interact property with Ap4A and its phosphonate analogues.     

Perindopril and ramipril phosphonate analogues as a new class of angiotensin converting enzyme inhibitors

A series of phosphonate analogues related to perindopril and ramipril were prepared and tested to estimate their ability to inhibit angiotensin converting enzyme. These new synthesized compounds were active ACE inhibitors with a promising activity.     

Increase in fidelity of rat liver Ile-tRNA formation by both spermine and the aminoacyl-tRNA synthetase complex

To examine the polyamine effects on the fidelity at the aminoacylation level and the physiological significance of the existence of the aminoacyl-tRNA synthetase complex (ARSC) in animal cells, a single-chain Ile-tRNA synthetase (IRSS) was isolated from the complex by treatment with trypsin. Ile-tRNA formation by IRSS was strongly stimulated by spermine, similar to the results with ARSC. Two misacylations (Val-tRNAIle and Ile-tRNAiMet formation) by IRSS were measured. The error frequency was higher in Ile-tRNAiMet formation (tRNA misacylation) than in Val-tRNAIle formation (amino acid misacylation). Spermine did not influence significantly Ile-tRNAiMet formation, but it stimulated Val-tRNAIle formation by IRSS. Accordingly, spermine decreased the error frequency of tRNA misacylation, but not amino acid misacylation. These results suggest that the conformational changes of individual tRNA by spermine differ from each other, meaning that spermine influences the interaction between individual tRNA and aminoacyl-tRNA synthetase variously. When the aminoacylations of tRNAIle from rat liver, yeast, and Escherichia coli were compared with ARSC and IRSS, the relative speed of Ile-tRNA formation with tRNAIle from other species was faster with IRSS than with ARSC. This indicates that ARSC can recognize tRNAIle from the same species more specifically than IRSS. These results show that both spermine and ARSC are involved in the increase of fidelity of rat liver Ile-tRNA formation.     

Adenosine 5'-tetraphosphate is synthesized by the histidine alpha 142----asparagine mutant of Escherichia coli succinyl-CoA synthetase.

Recently, we described the properties of a mutant (H142N) of Escherichia coli succinyl coenzyme A (CoA) synthetase in which His-142 of the alpha-subunit was changed to Asn (Luo, G.-X., and Nishimura, J.S. (1991) J. Biol. Chem. 266, 20781-20785). The mutant enzyme was practically devoid of ability to catalyze the overall reaction but was able to catalyze half-reactions at significant rates. Thus, phosphorylation by ATP and dephosphorylation by ADP of the mutant enzyme occurred at rates that were at least 10 times greater than those with wild type enzyme, and dephosphorylation by succinate plus CoA (succinyl-CoA formation) proceeded with a Vmax of 10% that of wild type, with no change in Km for succinate and very little change in Km for CoA. In the present work, it has been shown that incubation of 32P-labeled H142N with ATP caused a rapid depletion of label from the enzyme and incorporation of radioactivity into a nucleotide species that was neither ATP nor ADP. This reaction was catalyzed at comparatively negligible rates by wild type enzyme. Analysis of the labeled product by high pressure liquid chromatography and 31P NMR revealed that it was adenosine 5'-tetraphosphate (AP4). Incubation of labeled H142N with the ATP analog beta,gamma-methylene adenosine triphosphate also gave a product that appeared to be the corresponding tetraphosphate. The reaction in which AP4 was formed was greatly stimulated by the addition of phosphoenolpyruvate plus pyruvate kinase and strongly inhibited by ADP and by CoA plus succinate. The results are consistent with binding of ATP to, and reaction with, phosphorylated succinyl-CoA synthetase to form AP4. In this reaction, it was determined that the Km for ATP and the turnover number of phosphorylated enzyme were 14.5 microM and 0.024 s-1, respectively.     

Synthesis of glutaminyl adenylate analogues that are inhibitors of glutaminyl-tRNA synthetase

Glutaminol adenylate 5 is a competitive inhibitor of glutaminyl-tRNA synthetase with respect to glutamine (Ki = 280 nM) and to ATP (Ki = 860 nM). The corresponding methyl phosphate ester 4 is a weaker inhibitor (Ki approximately 10 microM) with respect to glutamine.     

A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA^Pro. The most important functional element of this catalytic mechanism is the 2′-OH group of the terminal adenosine 76 of Ala-tRNA^Pro, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3′-O atom of A76.     

Deinococcus glutaminyl-tRNA synthetase is a chimer between proteins from an ancient and the modern pathways of aminoacyl-tRNA formation

Glutaminyl-tRNA synthetase from Deinococcus radiodurans possesses a C-terminal extension of 215 residues appending the anticodon-binding domain. This domain constitutes a paralog of the Yqey protein present in various organisms and part of it is present in the C-terminal end of the GatB subunit of GatCAB, a partner of the indirect pathway of Gln-tRNA^Gln formation. To analyze the peculiarities of the structure–function relationship of this GlnRS related to the Yqey domain, a structure of the protein was solved from crystals diffracting at 2.3Å and a docking model of the synthetase complexed to tRNA^Gln constructed. The comparison of the modeled complex with the structure of the E. coli complex reveals that all residues of E. coli GlnRS contacting tRNA^Gln are conserved in D. radiodurans GlnRS, leaving the functional role of the Yqey domain puzzling. Kinetic investigations and tRNA-binding experiments of full length and Yqey-truncated GlnRSs reveal that the Yqey domain is involved in tRNA^Gln recognition. They demonstrate that Yqey plays the role of an affinity-enhancer of GlnRS for tRNA^Gln acting only in cis. However, the presence of Yqey in free state in organisms lacking GlnRS, suggests that this domain may exert additional cellular functions.