(1/70) The aroC gene of Aspergillus nidulans codes for a monofunctional, allosterically regulated chorismate mutase.
The cDNA and the chromosomal locus of the aroC gene of Aspergillus nidulans were cloned and is the first representative of a filamentous fungal gene encoding chorismate mutase (EC 184.108.40.206), the enzyme at the first branch point of aromatic amino acid biosynthesis. The aroC gene complements the Saccharomyces cerevisiae aro7Delta as well as the A. nidulans aroC mutation. The gene consists of three exons interrupted by two short intron sequences. The expressed mRNA is 0.96 kilobases in length and aroC expression is not regulated on the transcriptional level under amino acid starvation conditions. aroC encodes a monofunctional polypeptide of 268 amino acids. Purification of this 30-kDa enzyme allowed determination of its kinetic parameters (k(cat) = 82 s(-1), n(H) = 1. 56, [S](0.5) = 2.3 mM), varying pH dependence of catalytic activity in different regulatory states, and an acidic pI value of 4.7. Tryptophan acts as heterotropic activator and tyrosine as negative acting, heterotropic feedback-inhibitor with a K(i) of 2.8 microM. Immunological data, homology modeling, as well as electron microscopy studies, indicate that this chorismate mutase has a dimeric structure like the S. cerevisiae enzyme. Site-directed mutagenesis of a crucial residue in loop220s (Asp(233)) revealed differences concerning the intramolecular signal transduction for allosteric regulation of enzymatic activity. (+info)
(2/70) Characterization of hydroxylaminobenzene mutase from pNBZ139 cloned from Pseudomonas pseudoalcaligenes JS45. A highly associated SDS-stable enzyme catalyzing an intramolecular transfer of hydroxy groups.
Hydroxylaminobenzene mutase is the enzyme that converts intermediates formed during initial steps in the degradation of nitrobenzene to a novel ring-fission lower pathway in Pseudomonas pseudoalcaligenes JS45. The mutase catalyzes a rearrangement of hydroxylaminobenzene to 2-aminophenol. The mechanism of the reactions and the properties of the enzymes are unknown. In crude extracts, the hydroxylaminobenzene mutase was stable at SDS concentrations as high as 2%. A procedure including Hitrap-SP, Hitrap-Q and Cu(II)-chelating chromatography was used to partially purify the enzyme from an Escherichia coli clone. The partially purified enzyme was eluted in the void volume of a Superose-12 gel-filtration column even in the presence of 0.05% SDS in 25 mM Tris/HCl buffer, which indicated that it was highly associated. When the enzymatic conversion of hydroxylaminobenzene to 2-aminophenol was carried out in 18O-labeled water, the product did not contain 18O, as determined by GC-MS. The results indicate that the reaction proceeded by intramolecular transfer of the hydroxy group from the nitrogen to the C-2 position of the ring. The mechanism is clearly different from the intermolecular transfer of the hydroxy group in the non-enzymatic Bamberger rearrangement of hydroxylaminobenzene to 4-aminophenol and in the enzymatic hydroxymutation of chorismate to isochorismate. (+info)
(3/70) Archaeal shikimate kinase, a new member of the GHMP-kinase family.
Shikimate kinase (EC 220.127.116.11) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 18.104.22.168) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: K(m,shikimate) = 414 +/- 33 microM, K(m,ATP) = 48 +/- 4 microM, and k(cat) = 57 +/- 2 s(-1) for the predicted shikimate kinase and K(m,homoserine) = 188 +/- 37 microM, K(m,ATP) = 101 +/- 7 microM, and k(cat) = 28 +/- 1 s(-1) for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes. (+info)
(4/70) Essential PchG-dependent reduction in pyochelin biosynthesis of Pseudomonas aeruginosa.
The biosynthetic genes pchDCBA and pchEF, which are known to be required for the formation of the siderophore pyochelin and its precursors salicylate and dihydroaeruginoate (Dha), are clustered with the pchR regulatory gene on the chromosome of Pseudomonas aeruginosa. The 4.6-kb region located downstream of the pchEF genes was found to contain three additional, contiguous genes, pchG, pchH, and pchI, probably forming a pchEFGHI operon. The deduced amino acid sequences of PchH and PchI are similar to those of ATP binding cassette transport proteins with an export function. PchG is a homolog of the Yersinia pestis and Y. enterocolitica proteins YbtU and Irp3, which are involved in the biosynthesis of yersiniabactin. A null mutation in pchG abolished pyochelin formation, whereas mutations in pchH and pchI did not affect the amounts of salicylate, Dha, and pyochelin produced. The pyochelin biosynthetic genes were expressed from a vector promoter, uncoupling them from Fur-mediated repression by iron and PchR-dependent induction by pyochelin. In a P. aeruginosa mutant lacking the entire pyochelin biosynthetic gene cluster, the expressed pchDCBA and pchEFG genes were sufficient for salicylate, Dha, and pyochelin production. Pyochelin formation was also obtained in the heterologous host Escherichia coli expressing pchDCBA and pchEFG together with the E. coli entD gene, which provides a phosphopantetheinyl transferase necessary for PchE and PchF activation. The PchG protein was purified and used in combination with PchD and phosphopantetheinylated PchE and PchF in vitro to produce pyochelin from salicylate, L-cysteine, ATP, NADPH, and S-adenosylmethionine. Based on this assay, a reductase function was attributed to PchG. In summary, this study completes the identification of the biosynthetic genes required for pyochelin formation from chorismate in P. aeruginosa. (+info)
(5/70) The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan.
The crystal structure of anthranilate synthase (AS) from Serratia marcescens, a mesophilic bacterium, has been solved in the presence of its substrates, chorismate and glutamine, and one product, glutamate, at 1.95 A, and with its bound feedback inhibitor, tryptophan, at 2.4 A. In comparison with the AS structure from the hyperthermophile Sulfolobus solfataricus, the S. marcescens structure shows similar subunit structures but a markedly different oligomeric organization. One crystal form of the S. marcescens enzyme displays a bound pyruvate as well as a putative anthranilate (the nitrogen group is ambiguous) in the TrpE subunit. It also confirms the presence of a covalently bound glutamyl thioester intermediate in the TrpG subunit. The tryptophan-bound form reveals that the inhibitor binds at a site distinct from that of the substrate, chorismate. Bound tryptophan appears to prevent chorismate binding by a demonstrable conformational effect, and the structure reveals how occupancy of only one of the two feedback inhibition sites can immobilize the catalytic activity of both TrpE subunits. The presence of effectors in the structure provides a view of the locations of some of the amino acid residues in the active sites. Our findings are discussed in terms of the previously described AS structure of S. solfataricus, mutational data obtained from enteric bacteria, and the enzyme's mechanism of action. (+info)
(6/70) Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris.
Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA(II) proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream. (+info)
(7/70) The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.
BACKGROUND: Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. RESULTS: The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. CONCLUSIONS: Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously. (+info)
(8/70) Clustering of isochorismate synthase genes menF and entC and channeling of isochorismate in Escherichia coli.
There are two isochorismate synthase genes entC and menF in Escherichia coli. They encode enzymes (isochorismate synthase, EC 22.214.171.124) which reversibly synthesize isochorismic acid from chorismic acid. The genes share a 24.2% identity but are differently regulated. Activity of the MenF isochorismate synthase is significantly increased under anaerobic conditions whereas the activity of the EntC isochorismate synthase is greatly stimulated during growth in an iron deficient medium. Isochorismic acid synthesized by EntC is mainly channeled into enterobactin synthesis whereas isochorismic acid synthesized by MenF is mainly channeled into menaquinone synthesis. When menF or entC were separately placed onto overexpression plasmids and the plasmids introduced into a menF(-)/entC(-) double mutant in two separate experiments, the isochorismate formed was fed into both, the menaquinone and the enterobactin pathway. Moreover, in spite of a high isochorismate synthase activity menaquinone and enterobactin formation were not fully restored, indicating that isochorismate was lost by diffusion. Thus, under these conditions channeling was not observed. We conclude that in E. coli the chromosomal position of both menF and entC in their respective clusters is a prerequisite for channeling of isochorismate in both pathways. (+info)