Chloramphenicol Binding Site Research Articles

Investigation of radezolid interaction with non-canonical chloramphenicol binding site by molecular dynamics simulations

Radezolid is a promising antibiotic of oxazolidinone family, which is able to overcome effect of some linezolid resistance mechanisms of bacterial ribosomes. The structure of the radezolid complex with ribosomes was never published but, by analogy with linezolid, it is considered to prevent the binding of aminoacyl–tRNA to the A–site of the ribosome large subunit. However, as with linezolid, it can be assumed that radezolid binds to the alternative binding site existing in the A,A/P,P–ribosome. In the present article we have investigated this issue by molecular dynamics simulations and proposed the structure of the radezolid complex with a E. coli ribosome, which is consistent with available data of biochemical investigations of radezolid action.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

SHAPE footprinting as complementary approach to structure‐based design of ribosomal antibiotics: Phenicol antibiotics prevent A2451 2′OH acylation

Early biochemical experiments suggested that phenyl propanoid antibiotics, particularly chloramphenicol (CAM), had more than one binding site on ribosomes. Indeed recent X‐ray structures have shown two distinct, non‐overlapping, binding sites for CAM bound to 50S ribosomal subunits. The structure of CAM bound to the 50S ribosomal subunit of Haloarcula marismortui shows an overlap of the CAM binding site with that of macrolide antibiotics (e.g. erythromycin, etc). Whereas the structure of CAM bound to Deinococcus radiodurans 50S ribosomal subunits shows an overlaps between the binding site of CAM with that of puromycin or the CCA‐end of A‐site tRNA. We have used SHAPE footprinting to map the location, in solution, of antibiotics, including several novel phenicol analogs, onto 70S ribosomes from Escherichia coli and Staphylococcus aureus. Our data clearly indicate that phenyl propanoid antibiotics are able to simultaneously occupy both binding sites in E. coli as well as in S. aureus ribosomes. Nevertheless, "macrolide‐like" footprints were less reproducible than "puromycin‐like" (A‐site) footprints. We found that phenicol binding to the "puromycin" binding site blocks 2′‐OH hydroxyl modification of nucleotide A2451. Interestingly, this hydroxyl group has been shown to be critical in peptide bond formation, thus our results are shedding more light on the mechanism of phenyl propanoid antibiotic action.

Design, synthesis and ribosome binding of chloramphenicol nucleotide and intercalator conjugates

Molecular modelling based on X-ray structures of the antibiotic drug chloramphenicol bound in a bacterial ribosome has been used for design of chloramphenicol derivatives. Conjugates of the chloramphenicol amine through appropriate linkers to either a pyrene moiety or to a mono- or dinucleotide moiety were designed to improve binding to ribosomes by providing specific interactions in the peptidyl transferase site or to the P-loop in the ribosome. Specific binding of the conjugates were investigated by footprinting analysis using chemical modifications of accessible nucleotides in ribosomal RNA. The pyrene chloramphenicol conjugate shows enhanced binding to the chloramphenicol binding site compared to the native chloramphenicol, whereas the four nucleotide conjugates could not be shown to bind to the chloramphenicol binding site or to the P-loop.

A conserved chloramphenicol binding site at the entrance to the ribosomal peptide exit tunnel.

The antibiotic chloramphenicol produces modifications in 23S rRNA when bound to ribosomes from the bacterium Escherichia coli and the archaeon Halobacterium halobium and irradiated with 365 nm light. The modifications map to nucleotides m(5)U747 and C2611/C2612, in domains II and V, respectively, of E.coli 23S rRNA and G2084 (2058 in E.coli numbering) in domain V of H.halobium 23S rRNA. The modification sites overlap with a portion of the macrolide binding site and cluster at the entrance to the peptide exit tunnel. The data correlate with the recently reported chloramphenicol binding site on an archaeal ribosome and suggest that a similar binding site is present on the E.coli ribosome.

Steroid Recognition by Chloramphenicol Acetyltransferase: Engineering and Structural Analysis of a High Affinity Fusidic Acid Binding Site

The antibiotic fusidic acid and certain closely related steroidal compounds are potent competitive inhibitors of the type I variant of chloramphenicol acetyltransferase (CAT I). In the absence of crystallographic data for CAT I, the structural determinants of steroid binding were identified by (1) construction in vitroof genes encoding chimaeric enzymes containing segments of CAT Iand the related type III variant (CAT III) and (2) site-directed mutagenesis of the gene encoding CAT III, followed by kinetic characterisation of the substituted variants. Replacement of four residues of CAT III(Gln92, Asn146, Tyr168 and Ile172) by their equivalents from CAT Iyields an enzyme variant that is susceptible to competitive inhibition by fusidate with respect to chloramphenicol ( K i=5.4 μM). The structure of the complex of fusidate and the Q92C/N146F/Y168F/I172V variant, determined at 2.2 Å resolution by X-ray crystallography, reveals the inhibitor bound deep within the chloramphenicol binding site and in close proximity to the side-chain of His195, an essential catalytic residue. The aromatic side-chain of Phe146 provides a critical hydrophobic surface which interacts with non-polar substituents of the steroid. The remaining three substitutions act in concert both to maintain the appropriate orientation of Phe 146 and viaadditional interactions with the bound inhibitor. The substitution of Gln92 by Cys eliminates a critical hydrogen bond interaction which constrains a surface loop (residues 137 to 142) of wild-type CAT IIIwhich must move in order for fusidate to bind to the enzyme. Only two hydrogen bonds are observed in the CAT-fusidate complex, involving the 3-α-hydroxyl of the A-ring and both hydroxyl of Tyr25 and NE2 of His195, both of which are also involved in hydrogen bonds with substrate in the CAT III-chloramphenicol complex. In the acetyl transfer reaction catalysed by CAT, NE2, of His195 serves as a general base in the abstraction of a proton from the 3-hydroxyl of chloramphenicol as the first chemical step in catalysis. The structure of the CAT-inhibitor complex suggests that deprotonation of the 3-α-hydroxyl of bound fusidate by this mechanism could produce an oxyanion nucleophile analogous to that seen with chloramphenicol, but one which is incorrectly positioned to attack the thioester carbonyl of acetyl-CoA, accounting for the observed failure of CAT to acetylate fusidate.

Acetyl coenzyme A binding by chloramphenicol acetyltransferase: long-range electrostatic determinants of coenzyme A recognition.

The possible involvement of arginyl and lysyl side chains of chloramphenicol acetyltransferase (CAT) in binding coenzyme A (CoA) was studied by means of chemical modification, site-directed mutagenesis, variation in ionic strength, use of competitive inhibitors or substrate analogues, and X-ray crystallography. Unlike a number of enzymes, including citrate synthase, CAT does not employ specific ion pairs with the phosphoanionic centers of CoA to bind the acetyl donor, and arginyl residues play no role in recognition of the coenzyme. Although phenylglyoxal inactivates CAT reversibly, it does so by the formation of an unstable adduct with a thiol group, that of Cys-31 in the chloramphenicol binding site. The inhibitory effect of increasing ionic strength on kcat/Km(acetyl-CoA) can be explained by long-range electrostatic interactions between CoA and the epsilon-amino groups of Lys-54 and Lys-177, both of which are solvent-accessible. The epsilon-amino group of Lys-54 contributes 1.3 kcal.mol-1 to the binding of acetyl-CoA via interactions with both the 3'- and 5'-phosphoanions of CoA. Lys-177 contributes only 0.4 kcal.mol-1 to the productive binding of acetyl-CoA, mediated by long-range (approximately 14 A) interactions with the 5'-alpha- and -beta-phosphoanions of CoA. The combined energetic contribution of Lys-54 and Lys-177 to acetyl-CoA binding (1.7 kcal.mol-1) is less than that previously demonstrated (2.4 kcal.mol-1) for a simple hydrophobic interaction between Tyr-178 and the adenine ring of CoA (Day & Shaw, 1992).(ABSTRACT TRUNCATED AT 250 WORDS)

Stabilization of the imidazole ring of His-195 at the active site of chloramphenicol acetyltransferase

The imidazole of His-195 plays an essential role in the proposed general base mechanism of chloramphenicol acetyltransferase (CAT). The structure of the binary complex of CATIII and chloramphenicol suggests that two unusual interactions might determine the conformation of the side chain of His-195: (i) an intraresidue hydrogen bond between its main chain carbonyl and the protonated N delta 1 of the imidazole ring and (ii) face-to-face van der Waals contact between the His-195 imidazole group and the aromatic side chain of Tyr-25. Tyr-25 also makes a hydrogen bond, via its phenolic hydroxyl, to the carbonyl oxygen of the substrate chloramphenicol. Replacement of Tyr-25 of CATIII by phenylalanine results in a modest increase in the Km for chloramphenicol (from 11.6 to 14.6 microM) and a 2-fold fall in kcat (599 to 258 s-1), indicative of a free energy contribution to transition state binding of 0.6 kcal mol-1 for the hydrogen bond between Tyr-25 and chloramphenicol. In contrast, substitution of Tyr-25 by alanine yields an enzyme that is dramatically impaired in its ability to bind chloramphenicol (Km = 173 microM). As kcat for Ala-25 CAT is also reduced (130 s-1), the loss of the aryl group results in a 69-fold decrease in kcat/Km, corresponding to a free energy contribution to binding and catalysis of 2.5 kcal mol-1. In addition to the loss of the hydrogen bond between Tyr-25 and chloramphenicol, the loss of substrate affinity in Ala-25 CAT may be a direct consequence of reduced hydrophobicity of the chloramphenicol-binding site and/or the loss of critical constraints on the precise conformation of the catalytic imidazole. However, as with wild type CAT, inactivation of Ala-25 CAT by the affinity reagent 3-(bromoacetyl) chloramphenicol is accompanied by modification solely at N epsilon 2 of His-195. Hence, the results demonstrate that tautomeric stabilization of the imidazole ring persists in the absence of van der Waals interactions with the side chain of Tyr-25, probably as a consequence of hydrogen bonding between the protonated N delta 1 and the carbonyl oxygen of His-195.

Elimination of a reactive thiol group from the active site of chloramphenicol acetyltransferase

1. The type III variant of chloramphenicol acetyltransferase (CATIII) is resistant to inactivation by ionizable modifying reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and iodoacetate, whereas it is sensitive to inhibition by similar but uncharged reagents, including 4,4'-dithiodipyridine, methyl methanethiolsulphonate (MMTS) and iodoacetamide. The target for these thiol-modifying reagents has been postulated to be Cys-31. This residue is situated within a part of the chloramphenicol-binding site formed largely from the side chains of hydrophobic amino acid residues, which might be expected to discriminate against the access of ionized ligands to Cys-31. 2. The substitution of Cys-31 by alanine, serine, threonine or methionine yields an enzyme that is resistant to inactivation by thiol-specific reagents. Replacement of Cys-31 by alanine, serine or threonine results in increased Km values for chloramphenicol with only small changes in kcat.. In contrast, the Cys-31----Met substitution mainly affects kcat. values. Although the kcat. for chloramphenicol acetylation is decreased 13-fold compared with wild-type CAT, the kcat. for the acetyl-CoA hydrolysis reaction, which occurs in the absence of chloramphenicol, is increased 2.7-fold. 3. MMTS modification of cysteine residues results in an adduct (-CH2-S-S-CH3) that is structurally similar to the side chain of a methionine residue (-CH2-CH2-S-CH3). The kinetic properties of MMTS-modified CATIII closely resemble those of [Met31]CAT.

A new genetic polymorphism in the 16S ribosomal RNA gene of human motochondrial DNA

Sequence analysis of the 3' end of the 16S rRNA gene of mitochondrial DNA (mtDNA) revealed a single base change G----A, at position 3010. This mutation was first identified in a patient who had recovered from chloramphenicol-induced aplastic anaemia (CAP-induced AA). A link between this mutation and CAP-induced AA was ruled out by investigating three other similar patients, none of whom had the mutation. This mutation lies within or near the chloramphenicol binding site in a part of the 16S rRNA gene which shows high evolutionary conservation and where polymorphisms have not been previously reported. Hybridization tests with appropriate oligonucleotide probes in 114 individuals reveal that this mutation has a polymorphic frequency of about 14% in Europeans.

The importance of highly conserved nucleotides in the binding region of chloramphenicol at the peptidyl transfer centre of Escherichia coli 23S ribosomal RNA.

The peptidyl transfer site has been localized at the centre of domain V of 23S-like ribosomal RNA (rRNA) primarily on the basis of a chloramphenicol binding site. The implicated region constitutes an unstructured circle in the current secondary structural model which contains several universally conserved nucleotides. With a view to investigate the function of this RNA region further, four of these conserved nucleotides, including one indirectly implicated in chloramphenicol binding, were selected for mutation in Escherichia coli 23S rRNA using oligonucleotide primers. Mutant RNAs were expressed in vivo on a plasmid-encoded rRNA (rrnB) operon and each one yielded dramatically altered phenotypes. Cells exhibiting A2060----C or A2450----C transversions were inviable and it was shown by inserting the mutated genes after a temperature-inducible promoter that the mutant RNAs were directly responsible. In addition, a G2502----A transition caused a decreased growth rate, probably due to a partial selection against mutant ribosome incorporation into polysomes, while an A2503----C transversion produced a decreased growth rate and conferred resistance to chloramphenicol. All of the mutant RNAs were incorporated into 50S subunits, but while the two lethal mutant RNAs were strongly selected against in 70S ribosomes, the plasmid-encoded A2503----C RNA was preferred over the chromosome-encoded RNA, contrary to current regulatory theories. The results establish the critical structural and functional importance of highly conserved nucleotides in the chloramphenicol binding region. A mechanistic model is also presented to explain the disruptive effect of chloramphenicol (and other antibiotics) on peptide bond formation at the ribosomal subunit interface.

Chloramphenicol binding site of an fi − R-factor-specified variant of chloramphenicol acetyltransferase

Chloramphenicol acetyltransferase (EC 2.3.1.28) specified by the fi − R-factor (type II) is highly sensitive to sulfhydryl reagents. When this variant was treated with stoichiometric amounts of 2, 2′dithiobispyridine, 90% of the enzymatic activity was lost with concomitant introduction of 0.9to 1.0 thiopyridine groups per mole of enzyme protomer. In the presence of stoichiometric amounts of the substrate, chloramphenicol, the enzyme was neither inactivated nor modified by the sulfhydryl reagents. Acetyl-coenzyme A exerted no protective effects when present in the reaction mixture. The enzyme was also inactivated by cyanylation with a stoichiometric amount of 2-nitro-5-thiocyanobenzoic acid. Labeling native type II enzyme with iodo[ 14C]acetamide and subsequently subjecting it to peptic digestion yielded one radioactive peptide. This cysteine-containing peptide had the same sequence as that found near the cysteine close to the chloramphenicol binding site of the commonly occurring type 1 enzyme. In conclusion, this cysteine residue is essential for the catalytic activity of both types of enzyme and is located in or near the chloramphenicol binding site. It also seems that the cysteine in type II is more sensitive to sulfhydryl reagents than the homologous cysteine in type I, probably because it is more available for modification.

The Reactivity of Sulfhydryl Groups at the Active Site of an R-Factor -Specified Variant of Chloramphenicol Acetyltransferase

1. The commonly observed type I variant of chloramphenicol acetyltransferase specified by R factors is a tetrameric enzyme and contains four half-cysteine residues per identical subunit. All four thiols are reactive when the protein has been reduced in the presence of 6 M guanidine-HCl. 2. Iodoacetamide is an effective inhibitor of native chloramphenicol acetyltransferase. The incubation of iodo[14C]acetamide with enzyme leads to amido-carboxymethylation. Chloramphenicol is effective in preventing both inactivation and chemical modification, whereas acetyl-CoA fails to protect against either effect. Evidence is presented in support of the preferential reactivity of a single cysteine residue (S1) towards iodoacetamide. Iodoacetate is not an effective inhibitor and fails to react with the native enzyme under conditions which permit inactivation and chemical modification by iodoacetamide. 3. The chromogenic disulfides 2,2′-dithio-bis(pyridine) and 4,4′-dithio-bis(pyridine) are more effective at low concentrations than iodoacetamide as inhibitors of chloramphenicol acetyltransferase. Inhibition and covalent modification of chloramphenicol acetyltransferase by 2,2′-dithio-bis(pyridine) can be prevented by chloramphenicol but not by acetyl-CoA. The same half-cysteine-containing sequence (S1), adduced to be the site of alkylation by iodoacetamide, appears to be uniquely susceptible to mixed disulfide formation with 2,2′-dithio-bis(pyridine). Cyanylation of the 2-thiopyridyl-inhibited enzyme does not lead to reactivation. 4. Chloramphenicol acetyltransferase is also susceptible to inhibition by N-ethylmaleimide, p-mercuribenzoate, and 1-fluoro-2,4-dinitrobenzene, and protection is afforded in each case by chloramphenicol. Dinitrophenylation of one or more cysteine residues is inferred by the reactivation observed by incubation of the inactivated enzyme with reduced thiols. 5. The reaction of iodo[14C]acetamide with native chloramphenicol acetyltransferase followed by proteolytic digestion yields radioactive peptides corresponding to only three of the four known half-cysteine-containing sequences. The results of competition and protection experiments suggest that the S1 sequence is the preferential site of attack by iodoacetamide or 2,2′-dithio-bis(pyridine) and that it is sufficiently close to the chloramphenicol binding site to be protected by that substrate. A thiol-containing sequence designated S3 has been judged to be implicated in the binding of acetyl-CoA on the basis of (a) protection against iodoacetamide labelling and inactivation by that substrate, and (b) probable covalent and selective modification of the S3 thiol by bromoacetyl-CoA. 6. Low but reproducible rates of enzyme inactivation occur when chloramphenicol acetyltransferase is incubated in the presence of a number of CoA-related compounds. The inactivation process is not related to the low rates of hydrolysis of acyl-CoA derivatives, which have been noted in the absence of chloramphenicol. There is no evidence to suggest a sequential (‘ping-pong’) reaction mechanism with the formation of a covalent enzyme-substrate intermediate.

Chloramphenicol binding site with analogues of chloramphenicol and puromycin.

The effect of a series of puromycin analogues and aminoacyl chloramphenicol derivatives on poly(U,C)-directed polyphenylalanine synthesis in an Escherichia coli cell-free system was examined. A comparison between the structures and activities of the puromycin and chloramphenicol analogues was made to examine the proposal that ribosomal binding sites for both antibiotics overlap. Our results suggest that the dichloroacetamido group in the chloramphenicol molecule does not correspond to the role of the aminoacyl moieties of either puromycin or aminoacyl transfer ribonucleic acid. These results comparing the structures and activities of puromycin and chloramphenicol analogues also seem inconsistent with a common binding site for the p-substituted phenyl moieties of the two antibiotics. Previous data have indicated that both sites are mutually affected by the prior binding of either antibiotic. Although it is possible that chloramphenicol and puromycin may have overlapping bindings sites, no common structural features between the two antibiotics are supported by our data.

Antibiotics as probes of ribosome structure: binding of chloramphenicol and erythromycin to polyribosomes; effect of other antibiotics.

Antibiotics were used as probes of ribosome topology and function. Studies of [(14)C]chloramphenicol and [(14)C]erythromycin binding to ribosomes and polyribosomes revealed the following features. The requirement of high K(+) concentration (150 mM) for [(14)C]chloramphenicol binding to NH(4)Cl-washed ribosomes resulted from the washing procedure. Neither native 70S ribosomes nor polyribosomes require K(+) greater than 30 mM for [(14)C]chloramphenicol binding. Whereas [(14)C]chloramphenicol binds to both ribosomes and polyribosomes, [(14)C]erythromycin binds essentially only to ribosomes. After removal of peptidyl-transfer ribonucleic acid (tRNA) from polyribosomes, [(14)C]erythromycin could then be bound. The effects of a number of antibiotics on [(14)C]chloramphenicol binding to ribosomes and polyribosomes was assessed. It was found that most of the macrolides (erythromycin, carbomycin, spiramycin III, niddamycin, oleandomycin, and tylosin) and streptogramins A and B (vernamycin A, PA114A, vernamycin Balpha, and PA114B) inhibited chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes, but not to polyribosomes. After removal of peptidyl-tRNA from polyribosomes, [(14)C]chloramphenicol binding was then inhibited. In contrast, sparsomycin and althiomycin inhibited chloram-phenicol binding to polyribosomes, but not to ribosomes. After removal of peptidyl-tRNA from polyribosomes, sparsomycin and althiomycin were then ineffective. The presence of peptidyl-tRNA on polyribosomes apparently is required for binding of sparsomycin and althiomycin, but prevents binding of most macrolides and streptogramins. The lincosaminides (lincomycin and celesticetin) and methymycin (a small macrolide) inhibited [(14)C]chloramphenicol binding to NH(4)Cl-washed and native 70S ribosomes best, but also inhibited the binding to polyribosomes. The amino nucleosides and other antibiotics tested do not seem to interact strongly with the major chloramphenicol-binding site. These results provide knowledge of the interrelationships between antibiotic and substrate ribosome binding sites which should eventually contribute to a map of ribosomal topology.

Identification of chloramphenicol-binding protein in Escherichia coli ribosomes by affinity labeling.

Monoiodoamphenicol, a synthetic analogue of chloramphenicol, has been shown by competition experiments with chloramphenicol and lincomycin to bind at the same site of 70S ribosomes as chloramphenicol. At - 2 degrees it forms a 1:1 complex with 70S ribosomes having a value of K (7.5 x 10(4) M(-1)) that is one order of magnitude lower than that of chloramphenicol. At 37 degrees , monoiodoamphenicol irreversibly inhibits the protein-synthesizing activity of E. coli ribosomes. It is shown that the analogue reacted preferentially with protein L16 of E. coli 70S ribosomes, and we therefore conclude that protein L16 belongs to the chloramphenicol-binding site of E. coli ribosomes. Since the chemically reactive group of monoiodoamphenicol resembles iodoacetamide, the reaction of E. coli 70S ribosomes with monoiodoamphenicol was compared to that with iodoacetamide. Iodoacetamide did not react with protein L16, but it predominantly reacted with proteins S18 of the 30S subunit. Furthermore, monoiodoamphenicol was reacted with E. coli ribosomal subunits. Isolated 50S subunits bound monoiodoamphenicol by about one order of magnitude less than 70S ribosomes. Again, protein L16 reacted with the affinity label. Monoiodoamphenicol reacted with protein S18 in isolated 30S subunits; it also bound to 70S ribosomes of Bacillus stearothermophilus, however, it did not bind irreversibly to these 70S ribosomes.