Chorismate mutase catalyzes a key step in the shikimate biosynthetic pathway towards phenylalanine and tyrosine. Curiously, the intracellular chorismate mutase of Mycobacterium tuberculosis (MtCM; Rv0948c) has poor activity and lacks prominent active-site residues. However, its catalytic efficiency increases 4100-fold on addition of DAHP synthase (MtDS; Rv2178c), another shikimate-pathway enzyme. The 2.35 Å crystal structure of the MtCM-MtDS complex bound to a transition-state analogue shows a central core formed by four MtDS subunits sandwiched between two MtCM dimers. Structural comparisons imply catalytic activation to be a consequence of the repositioning of MtCM active-site residues on binding to MtDS. The mutagenesis of the Cterminal extrusion of MtCM establishes conserved residues as part of the activation machinery. The chorismatemutase activity of the complex, but not of MtCM alone, is inhibited synergistically by phenylalanine and tyrosine. The complex formation thus endows the shikimate pathway of M. tuberculosis with an important regulatory feature. Experimental evidence suggests that such noncovalent enzyme complexes comprising an AroQ d subclass chorismate mutase like MtCM are abundant in the bacterial order Actinomycetales.

The preferred way of life of Pseudomonas aeruginosa is aerobic respiration (38). When aeration is reduced or absent and nitrate is present, P. aeruginosa obtains energy from nitrate respiration (14,44). When neither oxygen nor nitrate is available, arginine can be used as the sole energy source for motility and growth (48,62). In this case, the arginine deiminase pathway serves to degrade arginine to ornithine and carbamoylphosphate, which allows regeneration of ATP from ADP without respiration (41,62). Anaerobic growth on arginine requires a rich medium and extended incubation; colonies take 4 to 5 days to form. Under these conditions, arginine is converted quantitatively to omithine and hence does not serve as a carbon source (62). Good aeration inhibits nitrate uptake (21) and prevents induction of the nitrate respiration enzymes (12) and of the arginine deiminase pathway in P. aeruginosa (34). Aerobic conditions favor the utilization of arginine as a carbon source via the succinylarginine pathway (13). The molecular mechanisms involved in the switch from aerobic to anaerobic metabolism in P. aeruginosa are unknown.In Escherichia coli and other enteric bacteria, thefnr gene is essential for the expression of fumarate and nitrate respiration and many other anaerobic processes (26,47,54,57,61). Thefnr-dependent genes and operons have a conserved symmetrical sequence, the FNR box, upstream of their promoters. Mutations in the FNR box interfere with anaerobic induction (8,16,24,54). It is generally accepted that under anaerobic conditions the FNR protein acts as a transcriptional activator by binding to the FNR box; however, a physical interaction between FNR and the FNR box has not been demonstrated (9, 54, 59).We are interested in the regulation of anaerobic metabolism in P. aeruginosa, especially in the induction of the arginine deiminase pathway by oxygen limitation. for this pathway are organized as an operon (arcDABC), which encodes a transmembrane protein having arginine/ ornithine antiporter activity (arcD), arginine deiminase (arcA), catabolic ornithine carbamoyltransferase (arcB), and carbamate kinase (arcC) (5, 6, 9a, 30, 31, 62). Most P. aeruginosa mutants that cannot grow anaerobically on arginine (Arc-phenotype) have a defect in the arcDABC operon (43, 62). Recently, we found a mutant whose Arc-phenotype is due to a mutation lying outside the arc operon (19). This mutation had originally been obtained by van Hartingsveldt et al. (63,64) and designated nirD. It causes inability of P. aeruginosa to grow anaerobically on nitrate (Nar-phenotype) and loss of dissimilatory nitrite reductase activity. Other genes required for nitrate respiration are unlinked to nirD (63,64). Here we show that a chromosomal fragment of P. aeruginosa complements the nirD mutant for anaerobic growth on nitrate and arginine and also restores anaerobic induction of nitrate reductase and arginine deiminase in the mutant. We therefore propose that nirD should be renamed anr (for anaerobic regulation of arginine deiminase and ni...

The gene encompassing ORF Rv1885c with weak sequence similarity to AroQ chorismate mutases (CMs) was cloned from the genome of Mycobacterium tuberculosis and expressed in Escherichia coli. The gene product (*MtCM) complements a CM‐deficient E. coli strain, but only if produced without the predicted N‐terminal signal sequence typical of M. tuberculosis. The mature *MtCM, which was purified by exploiting its resistance to irreversible thermal denaturation, possesses high CM activity in vitro. The enzyme follows simple Michaelis–Menten kinetics, having a kcat of 50 s−1 and a Km of 180 µm (at 30 °C and pH 7.5). *MtCM was shown to be a dimer by analytical ultracentrifugation and size‐exclusion chromatography. Secondary‐structure prediction and CD spectroscopy confirmed that *MtCM is a member of the all‐α‐helical AroQ class of CMs, but it seems to have a topologically rearranged AroQ fold. Because CMs are normally intracellular metabolic enzymes required for the biosynthesis of phenylalanine and tyrosine, the existence of an exported CM in Gram‐positive M. tuberculosis is puzzling. The observation that homologs of *MtCM with a predicted export sequence are generally only present in parasitic or pathogenic organisms suggests that secreted CMs may have evolved to participate in some aspect of parasitism or pathogenesis yet to be unraveled.

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway, which is inducible under conditions of oxygen limitation and serves to generate ATP from arginine. The 5' end of arc mRNA extracted from anaerobically grown cells was determined by Si and primer extension mapping. The transcription initiation site was located upstream of the arcD gene and 41.5 bp downstream of the center of the sequence TTGAC....ATCAG. This sequence, termed the ANR box, is similar to the consensus FNR recognition site of Escherichia coli. Transcription of the arc operon in P. aeruginosa was strongly decreased by a deletion of the TTGAC half site or by a mutation in the anr gene, which is known to code for the FNR-like regulatory protein ANR. During a transition from aerobic to anaerobic growth conditions, the concentrations of arc mRNAs and the levels of the ArcD and ArcA proteins rose in a parallel fashion. Mutational analysis of the arc promoter region led to the conclusion that the distance between the ANR box and the -10 promoter region is important for promoter strength, whereas the -35 region does not appear to be critical for arc promoter function. These findings and previous results indicate that anaerobic induction of the arc operon occurs at the level of transcription and requires the ANR box in cis and the ANR protein in trans.To obtain metabolic energy, Pseudomonas aeruginosa preferentially uses aerobic respiration. Under oxygen-limiting conditions, in the presence of nitrate or nitrite, P. aeruginosa switches to anaerobic respiration (9,37). In the absence of terminal electron acceptors, arginine can be used as the sole energy source (52). Anaerobic degradation of arginine to ornithine produces 1 mol of ATP per mol of arginine and depends on the three enzymes of the arginine deiminase pathway: arginine deiminase (ADI), catabolic omithine carbamoyltransferase, and carbamate kinase (Fig.

No abstract