Characterization and sequence of Escherichia coli pabC, the gene encoding aminodeoxychorismate lyase, a pyridoxal phosphate-containing enzyme

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In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubi4, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubi4. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubi4. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychorismate -. 4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.Chorismate is the branch point precursor for the synthesis of many aromatic compounds in Escherichia coli. The seven major end products of chorismate anabolism are phenylalanine, tyrosine, tryptophan, ubiquinone, 4-aminobenzoate, menaquinone, and enterobactin. Chorismate itself undergoes five different conversions that result in those seven products. The tyrosine and phenylalanine pathways diverge following the isomerization of chorismate to prephenate, but two distinct, homologous chorismate mutase activities channel chorismate toward either phenylalanine or tyrosine (10). The menaquinone and enterobactin pathways diverge following the conversion of chorismate to 2-hydroxy-4-deoxychorismate (isochorismate) by isochorismate synthase (28,30). For 4-aminobenzoate synthesis, chorismate is first aminated to form 4-amino-4-deoxychorismate, which then undergoes ,-elimination of the enol-pyruvyl moiety to form 4-aminobenzoate (1,8,21,29). The first step in ubiquinone biosynthesis is the conversion of chorismate to 4-hydroxybenzoate by chorismate lyase (5, 15), in a reaction apparently similar to the second step of 4-aminobenzoate synthesis, catalyzed by 4-amino-4-deoxychorismate lyase. Finally, the first step in tryptophan synthesis is chorismate amination and p-elimination of the enol-pyruvyl group to form anthranilate, or 2-aminobenzoate (2).With the exception of the genes involved in ubiquinone biosynthesis, all of the E. coli genes encoding enzymes that use chorismate or 4-amino-4-deoxychorismate as substrates have been cloned and sequenced. These include pheA (chorismate mutase-prephenate dehydratase), tyrA (chorismate mutase-prephenate dehydrogenase) (10), entC (isochorismate synth...

Section: Discussionmentioning

confidence: 95%

mentioning

confidence: 99%

Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase

Nichols

Green

1992

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“…In P. aeruginosa, a two-step conversion of chorismate to salicylate has been demonstrated, with the PchA protein acting as an isochorismate synthase (i.e., a chorismate isomerase, responsible for moving the hydroxyl group from position 4 to position 2 on the chorismate ring) and the PchB protein acting as an isochorismate-pyruvate lyase, cleaving the pyruvate moiety from the ring (17,18). This is equivalent to the situation in PABA production in many bacteria, where the PabB protein facilitates chemical modification of the chorismate ring (in this case the addition of an amino group is provided by the amidotransferase PabA) but a separate protein, PabC, provides lyase activity (24,37). In contrast, in tryptophan biosynthesis, the TrpE protein is able to act as both ring-modifying enzyme (again adding an amino group, in this case provided by TrpG) and lyase (1,56).…”

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confidence: 99%

The Structure of MbtI from Mycobacterium tuberculosis , the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase

Harrison

Gårdenborg

et al. 2006

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria. Mycobacterium tuberculosis acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for Pseudomonas aeruginosa, or a single-step conversion catalyzed by a salicylate synthase, as with Yersinia enterocolitica. Here we present the structure of the enzyme MbtI (Rv2386c) from M. tuberculosis, solved by multiwavelength anomalous diffraction at a resolution of 1.8 Å, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg 2؉ for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from Y. enterocolitica and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in M. tuberculosis.

“…Given the instability of clones containing E. coli fabF, it would be interesting to determine whether the V. harveyi gene can complement E. coli fabF mutants and confer temperature regulation of cis-vaccenic acid synthesis (17). Downstream of fabF is pabC (nucleotides 2964 to 3776), encoding a 29.9-kDa protein 35% identical to E. coli aminodeoxychorismate lyase (10). This enzyme appears to be less conserved between the two species than those involved in fatty acid synthesis.…”

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confidence: 99%

Isolation of Vibrio harveyi acyl carrier protein and the fabG, acpP, and fabF genes involved in fatty acid biosynthesis

Shen

Byers

1996

We report the isolation of Vibrio harveyi acyl carrier protein (ACP) and cloning of a 3,973-bp region containing the fabG (encoding 3-ketoacyl-ACP reductase, 25.5 kDa), acpP (encoding ACP, 8.7 kDa), fabF (encoding 3-ketoacyl-ACP synthase II, 43.1 kDa), and pabC (encoding aminodeoxychorismate lyase, 29.9 kDa) genes. Predicted amino acid sequences were, respectively, 78, 86, 76, and 35% identical to those of the corresponding Escherichia coli proteins. Five of the 11 sequence differences between V. harveyi and E. coli ACP were nonconservative amino acid differences concentrated in a loop region between helices I and II.Acyl carrier protein (ACP) is a highly conserved fatty acid carrier essential for the synthesis of fatty acids, phospholipids, and other complex molecules in a variety of organisms. The prototypic ACP from Escherichia coli is a 9-kDa acidic protein (pI, 4.1) of 77 amino acids which carries fatty acids as thioester intermediates attached to a phosphopantetheine prosthetic group at 31). In addition to its major function as a carrier for activated acyl intermediates during fatty acid elongation, ACP serves as an acyl donor for the synthesis of phospholipids (25), lipid A (1), and protein toxins such as hemolysin (12). ACP or ACP-like proteins have also been implicated in the synthesis of polyketides (27), periplasmic glucans involved in osmotic adaptation in E. coli (26), cell-host signaling (NodF factors) in Rhizobium leguminosarum (9), and cell wall synthesis in gram-positive bacteria (11). Thus, in a typical bacterial cell as many as a dozen different enzymes, with overlapping acyl chain specificities, compete for a small pool of acyl-ACP. Consequently, variations in the structure of ACP could have a large influence on the metabolic fate of acyl groups.In bioluminescent bacteria such as Vibrio harveyi, ACP is also involved in supplying myristic acid for the synthesis of the myristaldehyde substrate for luciferase (4, 6). A soluble fatty acyl-ACP synthetase which may be responsible for reentry of myristic acid into fatty acid biosynthetic pathways and/or activation of exogenous fatty acids to acyl-ACP in V. harveyi (5, 28) has been isolated (29). The present investigation was initiated to determine whether additional or different roles of ACP in a bioluminescent bacterium might be associated with particular structural features of the protein.Purification of V. harveyi ACP. ACP was purified from wildtype V. harveyi B392 grown in complex medium containing 1% NaCl (28) by using a modification of the isopropanol extraction procedure and batch chromatography on DEAE-Sepharose described for E. coli ACP (24). Further purification included chromatography on Sephacryl S-200 and Waters DEAE-5PW columns. ACP concentration was monitored by conversion to 3 H-acyl-ACP with partially purified V. harveyi acyl-ACP synthetase (29). Final purification was 1,700-fold, with a typical yield of 60 mg of ACP per kilogram (wet weight) of V. harveyi cells. Purified V. harveyi ACP migrated as a single 20-kDa band on a sodium d...