The redundant aminotransferases in lysine and arginine synthesis and the extent of aminotransferase redundancy in Escherichia coli.

Aminotransferases can be redundant or promiscuous, but the extent and significance of these properties is not known in any organism, even in Escherichia coli. To determine the extent of redundancy, it was first necessary to identify the redundant aminotransferases in arginine and lysine synthesis, and then complement all aminotransferase-deficient mutants with genes for all aminotransferases. The enzymes with N-acetylornithine aminotransferase (ACOAT) activity in arginine synthesis were ArgD, AstC, GabT and PuuE; the major anaerobic ACOAT was ArgD. The major enzymes with N-succinyl-l,l-diaminopimelate aminotransferase (SDAP-AT) activity in lysine synthesis were ArgD, AstC, and SerC. Seven other aminotransferases, when overproduced, complemented the defect in a triple mutant. Lysine availability did not regulate synthesis of the major SDAP-ATs. Complementation analysis of mutants lacking aminotransferases showed that the SDAP-ATs and alanine aminotransferases were exceptionally redundant, and it is proposed that this redundancy may ensure peptidoglycan synthesis. An overview of all aminotransferase reactions indicates that redundancy and broad specificity are common properties of aminotransferases.

Putrescine catabolism is a metabolic response to several stresses in Escherichia coli.

Genes whose products degrade arginine and ornithine, precursors of putrescine synthesis, are activated by either regulators of the nitrogen-regulated (Ntr) response or σ(S) -RNA polymerase. To determine if dual control regulates a complete putrescine catabolic pathway, we examined expression of patA and patD, which specify the first two enzymes of one putrescine catabolic pathway. Assays of PatA (putrescine transaminase) activity and ß-galactosidase from cells with patA-lacZ transcriptional and translational fusions indicate dual control of patA transcription and putrescine-stimulated patA translation. Similar assays for PatD indicate that patD transcription required σ(S) -RNA polymerase, and Nac, an Ntr regulator, enhanced the σ(S) -dependent transcription. Since Nac activation via σ(S) -RNA polymerase is without precedent, transcription with purified components was examined and the results confirmed this conclusion. This result indicates that the Ntr regulon can intrude into the σ(S) regulon. Strains lacking both polyamine catabolic pathways have defective responses to oxidative stress, high temperature and a sublethal concentration of an antibiotic. These defects and the σ(S) -dependent expression indicate that polyamine catabolism is a core metabolic response to stress.

Pathway and enzyme redundancy in putrescine catabolism in Escherichia coli.

Putrescine as the sole carbon source requires a novel catabolic pathway with glutamylated intermediates. Nitrogen limitation does not induce genes of this glutamylated putrescine (GP) pathway but instead induces genes for a putrescine catabolic pathway that starts with a transaminase-dependent deamination. We determined pathway utilization with putrescine as the sole nitrogen source by examining mutants with defects in both pathways. Blocks in both the GP and transaminase pathways were required to prevent growth with putrescine as the sole nitrogen source. Genetic and biochemical analyses showed redundant enzymes for γ-aminobutyraldehyde dehydrogenase (PatD/YdcW and PuuC), γ-aminobutyrate transaminase (GabT and PuuE), and succinic semialdehyde dehydrogenase (GabD and PuuC). PuuC is a nonspecific aldehyde dehydrogenase that oxidizes all the aldehydes in putrescine catabolism. A puuP mutant failed to use putrescine as the nitrogen source, which implies one major transporter for putrescine as the sole nitrogen source. Analysis of regulation of the GP pathway shows induction by putrescine and not by a product of putrescine catabolism and shows that putrescine accumulates in puuA, puuB, and puuC mutants but not in any other mutant. We conclude that two independent sets of enzymes can completely degrade putrescine to succinate and that their relative importance depends on the environment.

Genetics and regulation of the major enzymes of alanine synthesis in Escherichia coli.

Genetic analysis of alanine synthesis in the model genetic organism Escherichia coli has implicated avtA, the still uncharacterized alaA and alaB genes, and probably other genes. We identified alaA as yfbQ. We then transferred mutations in several transaminase genes into a yfbQ mutant and isolated a mutant that required alanine for optimal growth. For cells grown with carbon sources other than pyruvate, the major alanine-synthesizing transaminases are AvtA, YfbQ (AlaA), and YfdZ (which we designate AlaC). Growth with pyruvate as the carbon source and multicopy suppression suggest that several other transaminases can contribute to alanine synthesis. Expression studies showed that alanine modestly repressed avtA and yfbQ but had no effect on yfdZ. The leucine-responsive regulatory protein (Lrp) mediated control by alanine. We purified YfbQ and YfdZ and showed that both are dimers with K(m)s for pyruvate within the intracellular range of pyruvate concentration.

Crystal structure of N-succinylarginine dihydrolase AstB, bound to substrate and product, an enzyme from the arginine catabolic pathway of Escherichia coli.

The ammonia-producing arginine succinyltransferase pathway is the major pathway in Escherichia coli and related bacteria for arginine catabolism as a sole nitrogen source. This pathway consists of five steps, each catalyzed by a distinct enzyme. Here we report the crystal structure of N-succinylarginine dihydrolase AstB, the second enzyme of the arginine succinyltransferase pathway, providing the first structural insight into enzymes from this pathway. The enzyme exhibits a pseudo 5-fold symmetric alpha/beta propeller fold of circularly arranged betabetaalphabeta modules enclosing the active site. The crystal structure indicates clearly that this enzyme belongs to the amidinotransferase (AT) superfamily and that the active site contains a Cys-His-Glu triad characteristic of the AT superfamily. Structures of the complexes of AstB with the reaction product and a C365S mutant with bound the N-succinylarginine substrate suggest a catalytic mechanism that consists of two cycles of hydrolysis and ammonia release, with each cycle utilizing a mechanism similar to that proposed for arginine deiminases. Like other members of the AT superfamily of enzymes, AstB possesses a flexible loop that is disordered in the absence of substrate and assumes an ordered conformation upon substrate binding, shielding the ligand from the bulk solvent, thereby controlling substrate access and product release.

Phosphorylation-independent dimer-dimer interactions by the enhancer-binding activator NtrC of Escherichia coli: a third function for the C-terminal domain.

The response regulator NtrC transcriptionally activates genes of the nitrogen-regulated (Ntr) response. Phosphorylation of its N-terminal receiver domain stimulates an essential oligomerization of the central domain. Deletion of the central domain reduces, but does not eliminate, intermolecular interactions as assessed by cooperative binding to DNA. To analyze the structural determinants and function of this central domain-independent as well as phosphorylation-independent oligomerization, we randomly mutagenized DNA coding for an NtrC without its central domain and isolated strains containing NtrC with defective phosphorylation-independent cooperative binding. The alterations were primarily localized to helix B of the C-terminal domain. Site-specific mutagenesis that altered surface residues of helix B confirmed this localization. The purified NtrC variants, with or without the central domain, were specifically defective in phosphorylation-independent cooperative DNA binding and had little defect, if any, on other functions, such as non-cooperative DNA binding. We propose that this region forms an oligomerization interface. Full-length NtrC variants did not efficiently repress the glnA-ntrBC operon when NtrC was not phosphorylated, which suggests that phosphorylation-independent cooperative binding sets the basal level for glutamine synthetase and the regulators of the Ntr response. The NtrC variants in these cells generally, but not always, supported wild-type growth in nitrogen-limited media and wild-type activation of a variety of Ntr genes. We discuss the differences and similarities between the NtrC C-terminal domain and the homologous Fis, which is also capable of intermolecular interactions.

The Escherichia coli gabDTPC operon: specific gamma-aminobutyrate catabolism and nonspecific induction.

Nitrogen limitation induces the nitrogen-regulated (Ntr) response, which includes proteins that assimilate ammonia and scavenge nitrogen. Nitrogen limitation also induces catabolic pathways that degrade four metabolically related compounds: putrescine, arginine, ornithine, and gamma-aminobutyrate (GABA). We analyzed the structure, function, and regulation of the gab operon, whose products degrade GABA, a proposed intermediate in putrescine catabolism. We showed that the gabDTPC gene cluster constitutes an operon based partially on coregulation of GabT and GabD activities and the polarity of an insertion in gabT on gabC. A DeltagabDT mutant grew normally on all of the nitrogen sources tested except GABA. The unexpected growth with putrescine resulted from specific induction of gab-independent enzymes. Nac was required for gab transcription in vivo and in vitro. Ntr induction did not require GABA, but various nitrogen sources did not induce enzyme activity equally. A gabC (formerly ygaE) mutant grew faster with GABA and had elevated levels of gab operon products, which suggests that GabC is a repressor. GabC is proposed to reduce nitrogen source-specific modulation of expression. Unlike a wild-type strain, a gabC mutant utilized GABA as a carbon source and such growth required sigma(S). Previous studies showing sigma(S)-dependent gab expression in stationary phase involved gabC mutants, which suggests that such expression does not occur in wild-type strains. The seemingly narrow catabolic function of the gab operon is contrasted with the nonspecific (nitrogen source-independent) induction. We propose that the gab operon and the Ntr response itself contribute to putrescine and polyamine homeostasis.

Regulation of carbon utilization by sulfur availability in Escherichia coli and Salmonella typhimurium.

Different pleiotropic transcriptional regulators are known to function in the coordination of regulons concerned with carbon, nitrogen, sulfur, phosphorus and iron metabolism, but how expression profiles of these different regulons are coordinated with each other is not known. The basis for the effects of cysB mutations on carbon utilization in Escherichia coli and Salmonella typhimurium was examined. cysB mutations affected the utilization of some carbon sources more than others and these effects could be partially, but not completely, reversed by the inclusion of cysteine or djenkolate in the growth medium. Assays of transport systems and enzymes concerned with glucitol and alanine utilization showed that these activities were depressed in cysB mutants relative to isogenic wild-type strains, and cysteine or djenkolate present in the growth media partially restored these activities. Using transcriptional fusions to the fdo (formate dehydrogenase) and gut (glucitol) operons, it was shown that decreased expression resulted from defects at the transcriptional level. Furthermore, the effects of loss of CysB were much less pronounced under conditions of catabolite repression than in the absence of a catabolite-repressing carbon source, and cAMP largely reversed the effect of the loss of CysB. Comparable effects were seen for E. coli lacZ gene expression under the control of its own native promoter, and sulfur limitation in a cysB mutant depressed net cAMP production in a cAMP phosphodiesterase mutant. Adenylate cyclase thus appears to be responsive to sulfur deprivation. These observations may have physiological significance allowing carbon and sulfur regulon coordination during the growth of enteric bacteria in response to nutrient availability.