The isolation of a peptide from the catalytic domain of <i>Bacillus stearothermophilus</i> tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase

McArdell, J E C; Bruton, Chris J.; Atkinson, Tony

doi:10.1042/bj2430701

Cited by 7 publications

(2 citation statements)

References 32 publications

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“…The observation that ATP, NADP ϩ , and NADPH each can be used to elute this enzyme suggests that Reactive Green 19 binds to a nucleotide binding site on the enzyme. Reactive dyes have been used to map the nucleotide binding sites for other enzymes (11,21,22,27,31). Therefore, based on our results, Reactive Green 19 may be useful to probe the nucleotide binding site(s) of the aryl aldehyde oxidoreductase.…”

Section: Discussionmentioning

confidence: 78%

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Rosazza

1997

J Bacteriol

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Microbial reductions of aromatic carboxylic acids, usually to their corresponding alcohols, have been observed with wholecell biotransformations by a number of microorganisms, including Actinomyces (17), Clostridium thermoaceticum (31), Aspergillus niger (2, 24), Corynespora melonis (2), Coriolus (2), Neurospora (3), Glomerella cingulata (29, 30), Gloeosporium laeticolor (30), and Nocardia (8, 18). In all reported cases, substrates contained an aromatic moiety, although not all directly attached to the carboxyl groups being reduced to the corresponding alcohols. When the carbon next to the carboxyl group was chiral, the reduction displayed different degrees of enantioselectivity (8,29,30). The enantioselective step of this reduction has never been studied. Gross and Zenk found that in Neurospora crassa, an aldehyde intermediate was formed as a carboxylic acid was reduced to an alcohol (13,14). The enzyme reducing aryl carboxylic acids to aldehydes was purified and identified as a monomeric protein with an apparent molecular mass of 120,000 Da (13). The reduction required ATP, Mg 2ϩ , and NADPH as cofactors (13). Further work showed that this enzyme catalyzed the initial reaction between an aromatic acid and ATP to form an acyl-AMP intermediate which was subsequently reduced by NADPH to form the aldehyde (Fig. 1) (12). This enzyme was named as an aryl aldehyde oxidoreductase (EC 1.2.1.30).Kato et al. showed that Nocardia asteroides JCM 3016 could transform benzoic acid to benzyl alcohol (18). The enzyme from this organism reduces benzoic acid to benzaldehyde with ATP, NADPH, and Mg 2ϩ as cofactors. The K m values for benzoic acid, NADPH, and ATP were 260, 6, and 23 nM, respectively. A number of mono-and multisubstituted benzoic acids can be reduced by the enzyme. This aryl aldehyde oxidoreductase can also catalyze the reduction of benzoyl-AMP by NADPH (19). The Nocardia enzyme was purified, and it was proposed that it reduced benzoate by the same mechanism as the Neurospora enzyme (12).In our laboratory, a strain of Nocardia (NRRL 5646) was found to efficiently reduce aryl carboxylic acids to give aldehydes and alcohols. With this organism, the substrate specificity for carboxylic acids appears to be significantly different than that for the enzyme reported by Kato et al. (19). Whole-cell reductions with Nocardia sp. strain NRRL 5646 were highly enantioselective with ibuprofen isomers as substrates (8). We now report a rapid purification method, the characterization of the enzyme, the enantioselectivity of the enzyme with two isomers of ibuprofen, and the range of reductions of a series of aryl carboxylic acid substrates. MATERIALS AND METHODS Materials.A Mono-Q anion exchanger, hydroxyapatite, and molecular weight standards for gel electrophoresis were purchased from Bio-Rad (Hercules, Calif.). Polyvinylidene difluoride membranes were from Applied Biosystems (Foster City, Calif.). (S)-(ϩ)-␣-Methyl-4-(2-methylpropyl)-benzeneacetic acid (ibuprofen) was from Aldrich (Milwaukee, Wis.). The (R)-(Ϫ) isomer of ibuprofe...

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Section: Discussionmentioning

confidence: 78%

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Rosazza

1997

J Bacteriol

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“…The modes of binding of dyes to biological macromolecules, including proteins, nucleic acids, polysaccharides and lipids, is not clear at the molecular level and examples of protein-dye complexes in the literature are rare. Most examples that are found are not general in nature but involve the substitution of a coenzyme by a dye (for examples, see McArdell et al, 1987;Wassarman & Lentz, 1971) or the intercalation of a dye, such as acriflavin or acridine, among the stacked bases of RNA or DNA (Kennard & Hunter, 1989).…”

Section: Introductionmentioning

confidence: 99%

Investigation into the binding of dyes within protein crystals

McPherson

Larson

2018

Acta Cryst Sect F

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It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal-dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m-cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.

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Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns

Birch¹,

O’Byrne²,

Booth³

et al. 2003

Proteomics

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The reliable identification and analysis of the low abundance proteins expressed by a cell remains a key challenge in the study of cellular proteomes. The analysis of low abundance proteins is a particular problem when using two-dimensional gel electrophoresis (2-DE) to resolve the cellular proteins since the technology is unable to display the wide dynamic range of protein levels typically synthesized by cells. We have investigated the use of reactive dye compounds for the enrichment of low abundance cellular proteins prior to analysis by 2-DE. The capacity of reactive dye compounds to bind specific protein species was used as the basis for a general chromatographic tool for protein enrichment. Six reactive dye compounds were investigated in detail for the analysis of Escherichia coli proteins. Whole bacterial cell lysates were passed down columns prepared with the reactive dye compounds. The bound proteins were eluted with 1.5 M NaCl and analyzed by 2-DE. Distinctive protein profiles were observed for the bound proteins recovered from the different reactive dye compounds. Selected proteins enriched by these methods were identified by peptide mass mapping. The enrichment procedure developed using reactive dye compounds were used to investigate acid-induced changes in the proteome of E. coli grown at either pH 7.0 or pH 5.8. Increased levels of expression were observed for a number of proteins (for example, GdhA, PanC, ProC, TkrA, EF-TS and YodA) were observed for E. coli grown at pH 5.8. Five identified proteins (AroG, FabI, GlyA, PurA and EF-Tu) showed reduced levels of synthesis for bacteria grown at pH 5.8 compared to pH 7.0. In the case of PanC and FabI the altered expression profiles were only reliably demonstrated using the enrichment protocols. One theme emerging from these data was that the expression of proteins concerned with one-carbon metabolism was perturbed at pH 5.8, which may point to a previously unrecognized affect of low pH stress on the physiology of E. coli cells. We conclude that the prefractionation of cell lysates on reactive dye columns will serve as a valuable generic tool for the analysis of low abundance proteins expressed by both prokaryotic and eukaryotic cells.

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The isolation of a peptide from the catalytic domain of Bacillus stearothermophilus tryptophyl-tRNA synthetase. The interaction of Brown MX-5BR with tyrosyl-tRNA synthetase

Cited by 7 publications

References 32 publications

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Purification, characterization, and properties of an aryl aldehyde oxidoreductase from Nocardia sp. strain NRRL 5646

Investigation into the binding of dyes within protein crystals

Enrichment of Escherichia coli proteins by column chromatography on reactive dye columns

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