Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

Viswanathan, V. K.; Green, Jacalyn M.; Nichols, Brian P.

doi:10.1128/jb.177.20.5918-5923.1995

Cited by 53 publications

(42 citation statements)

References 21 publications

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“…ABZ2 ENCODES YEAST AMINODEOXYCHORISMATE LYASE 2109 on May 13, 2018 by guest http://ec.asm.org/ dimers and inactive monomers could be the basis of a mechanism of activity regulation of this enzyme, which does not appear to be feedback regulated for PABA or folates (5,42), and neither does its expression seem to be subject to transcriptional control (our unpublished results). Although unlikely, the possibility still exists that the presence of Abz2p monomers in the enzyme preparation could be an artifact due to the presence of an N-terminal His 6 tag in the recombinant protein used in our analyses.…”

Section: Vol 6 2007mentioning

confidence: 99%

A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2 , the Gene Encoding a Fungal Aminodeoxychorismate Lyase

et al. 2007

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Large-scale phenotypic analyses have proved to be useful strategies in providing functional clues about the uncharacterized yeast genes. We used here a chemogenomic profiling of yeast deletion collections to identify the core of cellular processes challenged by treatment with the p-aminobenzoate/folate antimetabolite sulfanilamide. In addition to sulfanilamide-hypersensitive mutants whose deleted genes can be categorized into a number of groups, including one-carbon related metabolism, vacuole biogenesis and vesicular transport, DNA metabolic and cell cycle processes, and lipid and amino acid metabolism, two uncharacterized open reading frames (YHI9 and YMR289w) were also identified. A detailed characterization of YMR289w revealed that this gene was required for growth in media lacking p-aminobenzoic or folic acid and encoded a 4-amino-4-deoxychorismate lyase, which is the last of the three enzymatic activities required for p-aminobenzoic acid biosynthesis. In light of these results, YMR289w was designated ABZ2, in accordance with the accepted nomenclature. ABZ2 was able to rescue the p-aminobenzoate auxotrophy of an Escherichia coli pabC mutant, thus demonstrating that ABZ2 and pabC are functional homologues. Phylogenetic analyses revealed that Abz2p is the founder member of a new group of fungal 4-amino-4-deoxychorismate lyases that have no significant homology to its bacterial or plant counterparts. Abz2p appeared to form homodimers and dimerization was indispensable for its catalytic activity.

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Section: Vol 6 2007mentioning

confidence: 99%

A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2 , the Gene Encoding a Fungal Aminodeoxychorismate Lyase

et al. 2007

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“…1). In the first step, the PabA and PabB proteins cooperate to replace the hydroxyl group of chorismate with an amino group, yielding 4-amino-4-deoxychorismate (ADC) (4,9). PabA acts as a glutamine amidotransferase, supplying an amino group to PabB, which carries out the amination reaction.…”

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confidence: 99%

“…Secondly, PABA synthases contain an extra region between the PabA and PabB domains that could potentially carry a novel ADC lyase activity. Lastly, PabA and PabB are, respectively, homologous to TrpG and TrpE, which convert chorismate all of the way to the PABA analog anthranilate (o-aminobenzoate) without need of a lyase (9,15). It thus remains to be established whether PABA synthases are bifunctional (i.e., strictly equivalent to PabA and PabB) or trifunctional, having in addition ADC lyase activity.…”

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confidence: 99%

Folate synthesis in plants: The p -aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids

Basset

Quinlivan

Ravanel

et al. 2004

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

115

111

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It is not known how plants synthesize the p-aminobenzoate (PABA) moiety of folates. In Escherichia coli, PABA is made from chorismate in two steps. First, the PabA and PabB proteins interact to catalyze transfer of the amide nitrogen of glutamine to chorismate, forming 4-amino-4-deoxychorismate (ADC). The PabC protein then mediates elimination of pyruvate and aromatization to give PABA. Fungi, actinomycetes, and Plasmodium spp. also synthesize PABA but have proteins comprising fused domains homologous to PabA and PabB. These bipartite proteins are commonly called ''PABA synthases,'' although it is unclear whether they produce PABA or ADC. Genomic approaches identified Arabidopsis and tomato cDNAs encoding bipartite proteins containing fused PabA and PabB domains, plus a putative chloroplast targeting peptide. These cDNAs encode functional enzymes, as demonstrated by complementation of an E. coli pabA pabB double mutant and a yeast PABA-synthase deletant. The partially purified recombinant Arabidopsis protein did not produce PABA unless the E. coli PabC enzyme was added, indicating that it forms ADC, not PABA. The enzyme behaved as a monomer in size-exclusion chromatography and was not inhibited by physiological concentrations of PABA, its glucose ester, or folates. When the putative targeting peptide was fused to GFP and expressed in protoplasts, the fusion protein appeared only in chloroplasts, indicating that PABA synthesis is plastidial. In the pericarp of tomato fruit, the PabA-PabB mRNA level fell drastically as ripening advanced, but there was no fall in total PABA content, which stayed between 0.7 and 2.3 nmol⅐g ؊1 fresh weight.

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“…[10] Two of these strains (ΔpabA and ΔpabB) have mutations in the genes encoding the two subunits of aminodeoxychorismate synthase, which catalyzes the penultimate step in PABA biosynthesis. [11] The third mutant (ΔaroC) is missing the gene encoding key shikimate pathway enzyme chorismate synthase, and is unable to synthesize not only PABA but also the aromatic amino acids L-phenylalanine, L-tyrosine, and L-tryptophan, as well as the aromatic metabolites para-hydroxybenzoic acid (PHBA) and 2,3-hydroxybenzoic acid. [12] We confirmed that all three strains were unable to grow in PABA-free M9 glycerol minimal media and established each strain's inability to utilize substrate 1 in place of PABA.…”

Section: Author Manuscriptmentioning

confidence: 99%

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

2013

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Chemical manipulations of small molecule metabolites take place in all living systems and are essential for sustaining life through cellular metabolism. [1] The vast majority of these transformations are carried out by macromolecular, enzymatic catalysts. In contrast to the natural world, most chemical reactions employed in laboratory syntheses utilize nonbiological reagents and catalysts. [2] Although non-biological catalysts and reagents have been shown to function in the presence of cells, [3] the question of whether their reactivity can interface with cellular metabolism and influence biological function remains underexplored. We are interested in further developing this aspect of reaction methodology, which we term biocompatible chemistry: non-enzymatic chemical transformations that can be used to manipulate the structures of small molecules in the presence of living organisms. Successful integration of biocompatible chemistry with cellular metabolism would have implications for biotechnology as it could expand the capabilities of biotransformation in ways that are not possible using enzymes. Establishing a link between non-enzymatic catalysis and metabolism could also have potential implications for prebiotic chemistry, as it would suggest that primitive cells could have relied upon non-enzymatic transformations occurring in the surrounding environment to generate essential metabolites. [4] Here we describe biocompatible chemical reactions that interact with microbial metabolism and rescue auxotrophy through the production of essential metabolites. This experimental approach enables us to directly connect the growth of a living organism to the success of a non-biological chemical transformation. *Prof. E. P. Balskus, balskus@chemistry.harvard.edu, http://scholar.harvard.edu/balskus. Supporting information for this article is available on the WWW under http://www.angewandte.org. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptAuxotrophs are organisms that lack the ability to synthesize key nutrients required for growth and survival. [5] The necessary compounds must instead be obtained from external sources, typically the diet or metabolic activities of other organisms living in close proximity. The numerous examples of auxtotrophs in Nature include humans, who are unable to synthesize many essential vitamins and metabolites. It is possible to rescue auxotrophy in bacteria by transformation with genes encoding enzymes that can replace or complement the missing metabolic activity. Restoration of growth in this way constitutes an extremely powerful strategy for selection of evolved biomolecules in the context of directed evolution. [6] Recently, Hecht and co-workers demonstrated that it is possible to rescue the growth of E. coli auxotrophs using de novo protein scaffolds not found in natural systems. [7] Significantly, this result implies that catalysts other than native enzymes can replace key metabolic functions in living cells.Our interest in merging non-enzymat...

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Kinetic characterization of 4-amino 4-deoxychorismate synthase from Escherichia coli

Cited by 53 publications

References 21 publications

A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2 , the Gene Encoding a Fungal Aminodeoxychorismate Lyase

A Chemogenomic Screening of Sulfanilamide-Hypersensitive Saccharomyces cerevisiae Mutants Uncovers ABZ2 , the Gene Encoding a Fungal Aminodeoxychorismate Lyase

Folate synthesis in plants: The p -aminobenzoate branch is initiated by a bifunctional PabA-PabB protein that is targeted to plastids

Rescuing Auxotrophic Microorganisms with Nonenzymatic Chemistry

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