Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome

Lázaro, Ester; Broek, L. A. G. M. Van Den; San‐Félix, Ana; Ottenheijm, H. C. J.; Ballesta, Juan P. G.

doi:10.1016/0300-9084(91)90157-v

Cited by 10 publications

(9 citation statements)

References 28 publications

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“…Whereas all attempts to alter the modified uracil base, except for removal of the methyl group (Fig. 1), inactivated the drug, the sulfoxy group could be modified with no loss of inhibitory activity (9). Moreover, there is a direct correlation between the degree of hydrophobicity of this part of the molecule and the inhibitory potential of the drug (1).…”

Section: Resultsmentioning

confidence: 99%

“…1) and unidentified ribosomal proteins (9). Weak evidence also exists for a direct antibiotic-rRNA interaction because haloarchaeal mutants carrying single-site mutations at positions 2438, 2451, 2499, 2500, or 2584 (lack of modification) within the peptidyl transferase loop region of 23S rRNA (Escherichia coli numbering) exhibit enhanced sparsomycin resistance (10)(11)(12).…”

Section: Sparsomycin (mentioning

confidence: 99%

“…Phenol-alanine-sparsomycin (named according to ref. 9) was dissolved in 20 l 50% DMSO at 7 mM and was iodinated with [ 125 I]NaI (100 Ci, Amersham Pharmacia) by using a mild procedure (6,19). [ 125 I]Phenol-alanine-sparsomycin (50 M) was subsequently complexed to 70S ribosomes (40 pmol) in the presence of poly(U) (1 g͞pmol ribosome) and N-Ac-[ 14 C]Phe-tRNA (1.3 mol͞mol ribosomes) in 80 l of 20 mM Tris⅐HCl (pH 7.5), 50 mM NH 4 Cl, and 10 mM MgCl 2 for 20 min at 37°C, and half of the sample was irradiated at 365 nm as described above.…”

Section: Sparsomycin Crosslinking To Ribosomes From Differentmentioning

confidence: 99%

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Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

Porse

Kirillov²,

Awayez³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [ 125 I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink. Sparsomycin (Fig.

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Section: Resultsmentioning

confidence: 99%

Section: Sparsomycin (mentioning

confidence: 99%

Section: Sparsomycin Crosslinking To Ribosomes From Differentmentioning

confidence: 99%

See 1 more Smart Citation

Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

Porse

Kirillov²,

Awayez³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Binding studies with various sparsomycin derivatives revealed two important groups, at either end of a fairly rigid molecule (Fig. 6 A), that interact with the ribosome‐tRNA complex [137, 138]; an S‐CH 3 group and a methylated uracil residue that carries a pseudouridine‐like linkage to a peptide‐like grouping [139]. Although the modified uracil can potentially base pair with rRNA, in contrast to the results obtained for many other peptidyl transferase drugs, footprinting studies failed to reveal evidence of an rRNA binding site for the drug [51, 135].…”

Section: The Peptidyl‐transferase Antibioticsmentioning

confidence: 95%

Movement of the 3′‐end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics

et al. 1997

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Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the peptidyl transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3'-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3'-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the peptidyl transferase antibiotics.

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“…Several total syntheses of sparsomycin have been reported previously (14,20,25,26). The biological activity of sparsomycin is the result of its ability to inhibit the peptide bond-forming step of protein biosynthesis by interacting with the large ribosomal subunit in a poorly understood, complex manner (16)(17)(18)30). Although the clinical use of sparsomycin is precluded due to drug-related retinopathy, a number of more potent analogs have been prepared which may prove useful in cancer chemotherapy (16).…”

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confidence: 99%

Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin

Parry

Hoyt

1997

J Bacteriol

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Sparsomycin is an antitumor antibiotic produced by Streptomyces sparsogenes. Biosynthetic experiments have previously demonstrated that one component of sparsomycin is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid and (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid. An enzyme which catalyzes the conversion of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid to (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid has been purified 740-fold to homogeneity from S. sparsogenes. The molecular mass of the native and denatured enzyme was 87 kDa, indicating that the native enzyme is monomeric. The enzyme required NAD ؉ for activity but lacked rigid substrate specificity, since analogs of both NAD ؉ and 3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid could serve as substrates. The enzyme was very weakly inhibited by mycophenolic acid. Monovalent cations were required for activity, with potassium ions being the most effective. The enzyme exhibited sensitivity toward diethylpyrocarbonate and some thiol-directed reagents, and it was irreversibly inhibited by 6-chloropurine. The properties of the enzyme suggest it is mechanistically related to inosine-5-monophosphate dehydrogenase.Sparsomycin (Fig. 1A) is an unusual monooxo-dithioacetalcontaining antibiotic that is produced by Streptomyces sparsogenes var. sparsogenes (3) and Streptomyces cuspidosporus (12). Sparsomycin displays bacteriocidal activity against a wide variety of gram-negative and gram-positive bacteria (27) as well as members of the Archaebacteria (17), and it exhibits potent anticancer activity against a variety of tumors in vivo and against KB human epidermoid carcinoma cells in culture (27). Sparsomycin has also been shown to potentiate the activity of other anticancer agents, such as cisplatin (25). The structure of sparsomycin was elucidated in 1970 by Wiley and MacKellar (33), while the absolute configuration was deduced by Ottenheijm and coworkers in 1981 (26). Several total syntheses of sparsomycin have been reported previously (14,20,25,26). The biological activity of sparsomycin is the result of its ability to inhibit the peptide bond-forming step of protein biosynthesis by interacting with the large ribosomal subunit in a poorly understood, complex manner (16)(17)(18)30). Although the clinical use of sparsomycin is precluded due to drug-related retinopathy, a number of more potent analogs have been prepared which may prove useful in cancer chemotherapy (16). One of the novel components of the sparsomycin molecule is (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid (UCA). Biosynthetic experiments have demonstrated (28) that this compound is derived from L-tryptophan via the intermediacy of (E)-3-(4-oxo-6-methyl-5-pyrimidinyl)acrylic acid (PCA) (Fig. 1A) and that both PCA and UCA are incorporated into sparsomycin. This article describes the first purification and characterization of an enzyme associated with the sparsomycin biosynthetic pathway. This enzyme, UCA synthase, catalyzes the NAD ϩ ...

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Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome

Cited by 10 publications

References 28 publications

Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

Movement of the 3′‐end of tRNA through the peptidyl transferase centre and its inhibition by antibiotics

Purification and preliminary characterization of (E)-3-(2,4-dioxo-6-methyl-5-pyrimidinyl)acrylic acid synthase, an enzyme involved in biosynthesis of the antitumor agent sparsomycin

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