Biodegradative Arginine Decarboxylase Research Articles

Structural studies on the decameric S. typhimurium arginine decarboxylase (ADC): Pyridoxal 5′-phosphate binding induces conformational changes

Enteric pathogens such as Salmonella typhimurium colonize the human gut in spite of the lethal acidic pH environment (pH < 2.5) due to the activation of inducible acid tolerance response (ATR) systems. The pyridoxal 5′-phosphate (PLP)-dependent enzyme, biodegradative arginine decarboxylase (ADC, encoded by AdiA), is a component of an ATR system. The enzyme consumes a cytoplasmic proton in the process of arginine degradation to agmatine. Arginine-agmatine antiporter (AdiC) exchanges the product agmatine for arginine. In this manuscript, we describe the structure of Salmonella typhimurium ADC (StADC). The decameric structure assembled from five dimers related by a non crystallographic 5-fold symmetry represents the first apo-form of the enzyme. The structure suggests that PLP-binding is not a prerequisite for oligomerization. Comparison with E. coli ADC reveals that PLP-binding is accompanied by the movement and ordering of two loops (residues 150–159 and 191–197) and a few active site residues such as His256 and Lys257. A number of residues important for substrate binding are disordered in the apo-StADC structure indicating that PLP binding is important for substrate binding. Unlike the interactions between 5-fold related protomers, interactions that stabilize the dimeric structure are not pH dependent.

Plasmids bearing hfq and the hns-like gene stpA complement hns mutants in modulating arginine decarboxylase gene expression in Escherichia coli

Biodegradative arginine decarboxylase is inducible by acid and is derepressed in an hns mutant. Several plasmids from an Escherichia coli library that could complement the hns phenotype were characterized and placed into groups. One group includes plasmids that contain the hns gene and are considered true complements. Another group was found to carry the hfq gene, which encodes the host factor HF-1 for bacteriophage Q beta replication. Plasmids of the third group contain inserts that mapped at 60.2 min on the E. coli chromosome. We identified an open reading frame (stpA) with a deduced amino acid sequence showing more than 60% identity with the sequences of H-NS proteins from several species as being responsible for the hns complementing phenotype of the third group.

Effects of rpoA and cysB mutations on acid induction of biodegradative arginine decarboxylase in Escherichia coli.

For Escherichia coli, there have been more and more examples illustrating that the alpha subunit of RNA polymerase is directly involved in the activation of gene transcription by interaction with activator proteins. Because of the vital function of the alpha subunit in cell growth, only a limited number of mutations in its structural gene, rpoA, have been isolated. We obtained a number of these mutants and examined the effects of these mutations on the acid induction of adi and cad gene expression. Several mutations caused a small reduction in adi promoter activity at inducing pH. One mutation, rpoA341, essentially eliminated adi promoter activity, while it had little effect on the cad promoter. During the course of a separate study, we isolated a plasmid that enhanced adi expression. Further characterization of this plasmid showed that it contained cysB, the structural gene for the positive regulator for most cys operon genes. Introduction of a cysB mutation into an adi::lac fusion strain and beta-galactosidase assay studies of the resultant adi::lac cysB mutant established that a wild-type cysB gene was required for efficient acid induction of adi expression. These results suggest that a possible interaction between CysB and the alpha subunit of RNA polymerase is involved in activation of adi transcription.

Modulation of acid-induced amino acid decarboxylase gene expression by hns in Escherichia coli.

Biodegradative arginine decarboxylase and lysine decarboxylase, encoded by adi and cadA, respectively, are induced to maximal levels when Escherichia coli is grown anaerobically in rich medium at acidic pH. Mutants formed by transposon mutagenesis, namely, GNB725, GNB729, GNB88, GNB824, and GNB837, exhibited considerably elevated expression at pH 8.0 compared with the corresponding parental strain. Southern hybridization and chromosome mapping showed that the above mutants contained a transposon within the hns gene. Several plasmids from an E. coli library able to complement these mutants by restoring normal pH induction were independently isolated and were found to contain the hns gene. These results suggest a role for the DNA-binding protein H-NS in affecting the activation of these acid-induced genes.

Biosynthesis of polyamines in ornithine decarboxylase, arginine decarboxylase, and agmatine ureohydrolase deletion mutants of Escherichia coli strain K-12.

Escherichia coli K-12 mutants that carry deletions in their genes for ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) (speC), arginine decarboxylase (L-arginine carboxy-lyase, EC 4.1.1.19) (speA), and agmatine ureohydrolase (agmatinase or agmatine amidinohydrolase, EC 3.5.3.11) (speB) can still synthesize very small amounts of putrescine and spermidine. The putrescine concentration in these mutants was found to be 1/2500th that in spe+ cells. The pathway of putrescine synthesis appears to be through the biodegradative arginine decarboxylase, which converts arginine to agmatine, in combination with a low agmatine ureohydrolase activity--1/2000th that in spe+ strains. These results suggest that even such low levels of polyamines permit a low level of protein synthesis. Evidence is presented that the polyamine requirement for the growth of the polyamine-dependent speAB, speC deletion mutants, which are also streptomycin resistant, is not due to a decreased ability to synthesize polyamines.

DL-alpha-(Difluoromethyl)arginine: a potent enzyme-activated irreversible inhibitor of bacterial decarboxylases.

DL-alpha-(Difluoromethyl)arginine (RMI 71 897) is an irreversible inhibitor of both the biosynthetic and biodegradative arginine decarboxylases of Escherichia coli and of the biosynthetic arginine decarboxylases of Pseudomonas aeruginosa and Klebsiella pneumoniae. The Ki is close to 800 muM for the biosynthetic decarboxylase of E. coli and 140 muM for the biodegradative enzyme while the respective half-lives (t1/2) calculated for an infinite concentration of inhibitor are 1.0 and 2.1 min. The inhibitor also blocks the arginine decarboxylase activity of E. coli and Pseudomonas aeruginosa in vivo, indicating that the compound is transported into the cell. DL-alpha-Methylarginine (RMI 71 699) was found to be a competitive inhibitor of both arginine decarboxylases from E. coli. These results suggest that it may be possible to use an arginine decarboxylase inhibitor in conjunction with known inhibitors of ornithine decarboxylase to block all putrescine biosynthesis in prokaryotic cells and thus to study the effects of such inhibition in these organisms.