Design of new photoaffinity labels for ribosomal peptidyltransferase

Quiggle, Kevin; Wejrowski, M L; Chládek, Stanislav

doi:10.1021/bi00594a013

Cited by 11 publications

(6 citation statements)

References 25 publications

(46 reference statements)

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“…Similar experiments have been tried in other studies with puromycin or puromycin-like affinity labels. Thus, Quiggle et al (1978) were unsuccessful in their attempts to transfer /V-AcPhe to what was presumably photoincorporated 2,(3,)-0-(4-azido-L-phenylalanyl)adenosine. phenyl)-L-lysinyl]puromycin aminonucleoside (NAPLys-PANS), but, as we discuss elsewhere (Nicholson et al, 1982), it is possible that transfer was actually to noncovalently bound material.…”

Section: Discussionmentioning

confidence: 99%

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analog of puromycin. III. Evidence for the functional site specificity of labeling

Nicholson¹,

Hall²,

Strycharz³

et al. 1982

Biochemistry

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The photoincorporation of p-azido[3H]puromycin [6-(dimethylamino)-9-[3'-deoxy-3'-[(p-azido-L-phenylalanyl)amino]-beta-D-ribofuranosyl]purine] into specific ribosomal proteins and ribosomal RNA [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Cooperman, B. S. (1982) Biochemistry (preceding paper in this issue)] is decreased in the presence of puromycin, thus demonstrating that labeling is site specific. The magnitudes of the decreases in incorporation into the major labeled 50S proteins found on addition of different potential ribosome ligands parallel the abilities of these same ligands to inhibit peptidyltransferase. This result provides evidence that p-azidopuromycin photoincorporation into these proteins occurs at the peptidyltransferase center of the 50S subunit, a conclusion supported by other studies of ribosome structure and function. A striking new finding of this work is that puromycin aminonucleoside is a competitive inhibitor of puromycin in peptidyltransferase. The photoincorporation of p-azidopuromycin is accompanied by loss of ribosomal function, but photoincorporated p-azidopuromycin is not a competent peptidyl acceptor. The significance of these results is discussed. Photolabeling of 30S proteins by p-azidopuromycin apparently takes place from sites of lower puromycin affinity than that of the 50S site. The possible relationship of the major proteins labeled, S18, S7, and S14, to tRNA binding is considered.

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Section: Discussionmentioning

confidence: 99%

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analog of puromycin. III. Evidence for the functional site specificity of labeling

Nicholson¹,

Hall²,

Strycharz³

et al. 1982

Biochemistry

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“…The yields of these compounds were in the 10-20% range. The N-protecting groups were removed by hydrogenolysis in acetic acid or by trifiuoroacetic acid [for C-A-Met (Quiggle et al, 1978)].…”

Section: Methodsmentioning

confidence: 99%

Stereochemical control of ribosomal peptidyltransferase reaction. Role of amino acid side-chain orientation of acceptor substrate

Bhuta¹,

Quiggle²,

Ott³

et al. 1981

Biochemistry

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The substrate specificity of the acceptor site of peptidyltransferase of Escherichia coli 70S ribosomes was investigated in the fMet-tRNA.A-U-G.70S ribosome and AcPhe-tRNA.poly(U).70S ribosome systems by using a series of 2'- and 3'-aminoacyldinucleoside phosphates as acceptors. These chemically synthesized compounds are analogues of the 3' termini of either 2'(3')-, 2'-, or 3'-aminoacyl transfer ribonucleic acids (AA-tRNAs) of the types C-A-aa, C-2'-dA-aa, C-3'-dA-aa, C-3'-dA-3'-NH-aa, and C-2'-dA-2'-NH-aa (aa = Phe, D-Phe, Lys, Leu, Ala, Glu, Pro, Gly, Asp, Met, and alpha-aminoisobutyryl). It was found that the 3'-aminoacyl derivatives of optically active amino acids are much better acceptors of N-formyl-L-methionine (fMet) or N-acetyl-L-phenylalanine (AcPhe) residues than the isomeric 2'-aminoacyl derivatives with affinity constant ratios (KM 2'/3') greater than 100. Likewise, C-A(D-Phe) is a weaker acceptor than the corresponding L derivative C-A-Phe. In contrast, all glycyl derivatives (C-2'-dA-Gly, C-3'-dA-Gly, C-3'-dA-3'-NH-Gly and C-2'-dA-2'-NH-Gly) are good acceptors of the fMet residue, with ratios (KM 2'/3') of approximately 2. On the basis of these results, a model for the stereochemical control of the peptidyl-transferase reaction is proposed. It assigns a major role to the orientation of the amino acid side chain in 2'- or 3'-AA-tRNA. A detailed model of the interaction of the acceptor terminus of 3'-AA-tRNA with the acceptor site of peptidyltransferase is also proposed. The model is strikingly similar to those for the active sites of proteolytic enzymes.

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“…The deamination of N -6-methyladenine was followed directly at 270 nm (Δε = 15,000 M −1 cm −1 ) 8. The cleavage of methanol from 6-methoxypurine was monitored at 270 nm (Δε = 3760 M −1 cm −1 ) and the loss of methylthiol from 6-methylmercaptopurine was determined at 290 nm (Δε = 20,200 M −1 cm −1 ) 14,15. The formation of hypoxanthine was confirmed using UV absorbance spectroscopy.…”

Section: Enzymatic Assaysmentioning

confidence: 99%

Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine

Kamat

Fan²,

Sauder

et al. 2011

J. Am. Chem. Soc.

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Two enzymes of unknown function from the amidohydrolase superfamily were discovered to catalyze the deamination of N-6-methyladenine to hypoxanthine and methyl amine. The methylation of adenine in bacterial DNA is a common modification for the protection of host DNA against restriction endonucleases. The enzyme from Bacillus halodurans, Bh0637, catalyzes the deamination of N-6-methyladenine with a k cat of 185 s −1 and a k cat /K m of 2.5 × 10 6 M −1 s −1 . Bh0637 catalyzes the deamination of N-6-methyladenine two orders of magnitude faster than adenine. A comparative model of Bh0637 was computed using the three-dimensional structure of Atu4426 (PDB code: 3NQB) as a structural template and computational docking was used to rationalize the preferential utilization of N-6-methyladenine over adenine. This is the first identification of an N-6-methyladenine deaminase (6-MAD).NCBI has classified proteins from completely sequenced bacterial genomes that perform the same or similar functions into Clusters of Orthologous Groups (COG).1 One of these clusters, cog1001, consists of ~250 proteins, all of which are members of the amidohydrolase superfamily (AHS). The AHS is an ensemble of evolutionarily related enzymes capable of hydrolyzing amide, amine, or ester functional groups at carbon and phosphorus centers.2 Enzymes within this superfamily possess a mononuclear or binuclear metal center embedded within a (β/α) 8 -barrel structural fold. 2 Cog1001A sequence similarity network for cog1001 is presented in Figure 1 at an E-value cutoff of 10 −80 .3 ,4 Adenine deaminase (ADE) is the predominant enzyme in cog1001 and is represented in Figure 1 by subgroups 1 and 4. Subgroups 3 and 5 consist of enzymes that are * raushel@tamu.edu . Supporting Information Available:The procedures for the purification of Bh0637 and Bsu06560 are provided. Table S1 lists the proteins belonging to subgroup 2 of cog1001 and Chart S1 presents the structures of the compounds tested for catalytic activity. Scheme S1 provides a sequence alignment between Bh0637 and Atu4426. This material is available free of charge via the Internet at http://pubs.acs.org. The functional annotations for most members of subgroup 2 in Figure 1 are currently listed as unknown in the NCBI database but some of them have been labeled as adenine deaminases without apparent experimental validation. Proteins in subgroup 2 have less than 35% sequence identity to the prototypical adenine deaminases found in subgroups 1 and 4. NIH Public AccessThere are sequences available for ~32 proteins from subgroup 2. These enzymes are found in various species of Bacillus. At least a dozen of these microorganisms also have an authentic adenine deaminase from subgroup 1 in their genome. Most of the genes in subgroup 2 from cog1001 lie adjacent to the pur operon for the biosynthesis of purine nucleotides. Moreover, in Bacillus halodurans, the genes for ADE from subgroup 1 (Bh0640) and Bh0637 from subgroup 2 are nearly adjacent to one another. The colocalization of these two genes sugge...

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Design of new photoaffinity labels for ribosomal peptidyltransferase

Cited by 11 publications

References 25 publications

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analog of puromycin. III. Evidence for the functional site specificity of labeling

Photoaffinity labeling of Escherichia coli ribosomes by an aryl azide analog of puromycin. III. Evidence for the functional site specificity of labeling

Stereochemical control of ribosomal peptidyltransferase reaction. Role of amino acid side-chain orientation of acceptor substrate

Enzymatic Deamination of the Epigenetic Base N-6-Methyladenine

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