Spectrophotometric assay for lysine decar☐ylase

Phan, Alvin; Ngo, T. T.; Lenhoff, Howard M.

doi:10.1016/0003-2697(82)90336-0

Cited by 72 publications

(45 citation statements)

References 8 publications

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“…In contrast, cadaverine, a product of the decarboxylation of lysine, is secreted from LDC ϩ cells (13). We measured supernatants of BS529 grown in LB for the presence of cadaverine by using a quantitative spectrophotometric assay (17). Whereas cultures of wild-type S. flexneri 2a strain 2457T contained no measurable cadaverine, BS529 supernatants contained 225-300 M cadaverine.…”

Section: Resultsmentioning

confidence: 99%

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“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Maurelli

Fernández²,

Bloch³

et al. 1998

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

408

308

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Plasmids, bacteriophages, and pathogenicity islands are genomic additions that contribute to the evolution of bacterial pathogens. For example, Shigella spp., the causative agents of bacillary dysentery, differ from the closely related commensal Escherichia coli in the presence of a plasmid in Shigella that encodes virulence functions. However, pathogenic bacteria also may lack properties that are characteristic of nonpathogens. Lysine decarboxylase (LDC) activity is present in Ϸ90% of E. coli strains but is uniformly absent in Shigella strains. When the gene for LDC, cadA, was introduced into Shigella flexneri 2a, virulence became attenuated, and enterotoxin activity was inhibited greatly. The enterotoxin inhibitor was identified as cadaverine, a product of the reaction catalyzed by LDC. Comparison of the S. flexneri 2a and laboratory E. coli K-12 genomes in the region of cadA revealed a large deletion in Shigella. Representative strains of Shigella spp. and enteroinvasive E. coli displayed similar deletions of cadA. Our results suggest that, as Shigella spp. evolved from E. coli to become pathogens, they not only acquired virulence genes on a plasmid but also shed genes via deletions. The formation of these ''black holes,'' deletions of genes that are detrimental to a pathogenic lifestyle, provides an evolutionary pathway that enables a pathogen to enhance virulence. Furthermore, the demonstration that cadaverine can inhibit enterotoxin activity may lead to more general models about toxin activity or entry into cells and suggests an avenue for antitoxin therapy. Thus, understanding the role of black holes in pathogen evolution may yield clues to new treatments of infectious diseases.

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Section: Resultsmentioning

confidence: 99%

“…Assays for LDC activity were those of Falkow (16) and Phan et al (17). The former assay provides a qualitative measure of LDC activity based on a shift in pH from acid to alkaline due to the production of cadaverine from the decarboxylation of lysine.…”

mentioning

confidence: 99%

“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

Maurelli

Fernández²,

Bloch³

et al. 1998

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

408

308

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“…To each sample toluene (1 ml) was added, followed by vigorously mixing for 20 s. The toluene and aqueous layers were separated by centrifugation. N,NЈ-Bistrinitrophenyllysine is soluble in water but not in toluene, whereas the reverse is true for N,NЈ-bistrinitrophenyl cadaverine (21). The absorption of N,NЈ-bistrinitrophenyl cadaverine (toluene phase) was determined at 340 nm, and the specific activity of CadA (mol/min*mg) was calculated.…”

Section: Methodsmentioning

confidence: 99%

New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli

Haneburger

Eichinger

Skerra

et al. 2011

Journal of Biological Chemistry

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The membrane-integrated transcriptional regulator CadC of Escherichia coli activates expression of the cadBA operon at low external pH with concomitantly available lysine, providing adaptation to mild acidic stress. CadC is a representative of the ToxR-like proteins that combine sensory, signal transduction, and DNA-binding activities within a single polypeptide. Although several ToxR-like regulators such as CadC, as well as the main regulator of Vibrio cholerae virulence, ToxR itself, which activate gene expression at acidic pH, have been intensively investigated, their molecular activation mechanism is still unclear. In this study, a structure-guided mutational analysis was performed to elucidate the mechanism by which CadC detects acidification of the external milieu. Thus, a cluster of negatively charged amino acids (Asp-198, Asp-200, Glu-461, Glu-468, and Asp-471) was found to be crucial for pH detection. These amino acids form a negatively charged patch on the surface of the periplasmic domain of CadC that stretches across its two subdomains. The results of different combinations of amino acid replacements within this patch indicated that the N-terminal subdomain integrates and transduces the signals coming from both subdomains to the transmembrane domain. Alterations in the phospholipid composition did not influence pH-dependent cadBA expression, and therefore, interplay of the acidic surface patch with the negatively charged headgroups is unlikely. Models are discussed according to which protonation of these acidic amino acid side chains reduces repulsive forces between the two subdomains and/or between two monomers within a CadC dimer and thereby enables receptor activation upon lowering of the environmental pH.

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“…Determination of Lysine Decarboxylase Activity-Lysine decarboxylase activity was determined essentially as described by Phan et al (13).…”

Section: Methodsmentioning

confidence: 99%

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

Kruse

Gehl

Geisler

et al. 2010

Journal of Biological Chemistry

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The molybdenum cofactor (Moco) forms part of the catalytic center in all eukaryotic molybdenum enzymes and is synthesized in a highly conserved pathway. Among eukaryotes, very little is known about the processes taking place subsequent to Moco biosynthesis, i.e. Moco transfer, allocation, and insertion into molybdenum enzymes. In the model plant Arabidopsis thaliana, we identified a novel protein family consisting of nine members that after recombinant expression are able to bind The molybdenum cofactor (Moco)2 is a prosthetic group highly conserved in all kingdoms of life and consists of a tricyclic pterin, referred to as molybdopterin or metal-binding pterin (MPT) and a molybdenum (Mo) atom covalently bound to the dithiolate moiety of MPT (1). Moco is required for the activity of all Mo-dependent enzymes with the exception of nitrogenase (2). Molybdenum enzymes (Mo-enzymes) are essential for a broad variety of metabolic processes such as nitrate assimilation and phytohormone synthesis in plants (3) and sulfur detoxification and purine catabolism in mammals (4).Synthesis of Moco proceeds in a highly conserved multistep pathway, involving at least six proteins named Cnx in plants (3). Much is known about the final step of Moco biosynthesis where one Mo atom is ligated to the MPT dithiolate function, which is catalyzed by the two-domain protein Cnx1 (5, 6): the C-terminal Cnx1-G domain activates MPT by adenylation, which is handed over to the N-terminal Cnx1-E domain where it is converted to Moco by inserting Mo into MPT under simultaneous cleavage of the pyrophosphate bond.After completion of biosynthesis, Moco has to be allocated and inserted into the apoMo-enzymes. In prokaryotes, a complex of proteins synthesizing the last steps of Moco biosynthesis donates the mature cofactor to apoenzymes assisted by enzyme-specific chaperones (7). In eukaryotes, however, no Mo-enzyme-specific chaperone has been found. As free Moco is extremely sensitive to oxidation it is also assumed that Moco occurs permanently protein-bound in the cell. Therefore, a cellular Moco distribution system should meet two demands: (i) it should bind Moco subsequent to its synthesis, and (ii) it should maintain a directed flow of Moco from the Moco donor Cnx1-E to the Mo-dependent enzymes. This is important to ensure the fast and efficient incorporation of Moco into apoMo-enzymes. In the alga Chlamydomonas reinhardtii a Moco carrier protein (MCP) was identified that was found to bind Moco and protect it against oxidation (8 -10). Without any denaturing procedure, subsequent transfer of Moco from MCP to apo-nitrate reductase (NR) from Neurospora crassa mutant nit-1 was possible (10), thus indicating that MCP-bound Moco was readily transferable. These properties of Chlamydomonas MCP make it a promising candidate for being part of a cellular Moco delivery system. It is, however, unknown whether MCP is also able to donate Moco to Mo-enzymes other than NR.Here we present the cloning and characterization of Mocobinding proteins (MoBP)

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Spectrophotometric assay for lysine decar☐ylase

Cited by 72 publications

References 8 publications

“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

“Black holes” and bacterial pathogenicity: A large genomic deletion that enhances the virulence of Shigella spp. and enteroinvasive Escherichia coli

New Insights into the Signaling Mechanism of the pH-responsive, Membrane-integrated Transcriptional Activator CadC of Escherichia coli

Identification and Biochemical Characterization of Molybdenum Cofactor-binding Proteins from Arabidopsis thaliana

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