Loss of allosteric control but retention of the bifunctional catalytic competence of a fusion protein formed by excision of 260 base pairs from the 3' terminus of pheA from Erwinia herbicola.
ABSTRACT: A bifunctional protein denoted as the P protein and encoded by pheA is widely present in purple gram-negative bacteria. This P protein carries catalytic domains that specify chorismate mutase (CM-P) and prephenate dehydratase. The instability of a recombinant plasmid carrying a pheA insert cloned from Erwinia herbicola resulted in a loss of 260 bp plus the TAA stop codon from the 3' terminus of pheA. The plasmid carrying the truncated pheA gene (denoted pheA*) was able to complement an Escherichia coli pheA auxotroph. pheA* was shown to be a chimera composed of the residual 5' part of pheA (901 bp) and a 5-bp fragment from the pUC18 vector. The new fusion protein (PheA*) retained both chorismate mutase and prephenate dehydratase activities. PheA* had a calculated subunit molecular weight of 33,574, in comparison to the 43,182-molecular-weight subunit size of PheA. The deletion did not affect the ability of PheA* to assume the native dimeric configuration of PheA. Both the CM-P and prephenate dehydratase components of PheA* were insensitive to L-phenylalanine inhibition, in contrast to the corresponding components of PheA. L-Phenylalanine protected both catalytic activities of PheA from thermal inactivation, and this protective effect of L-phenylalanine upon the PheA* activities was lost. PheA* was more stable than PheA to thermal inactivation; this was more pronounced for prephenate dehydratase than for CM-P. In the presence of dithiothreitol, the differential resistance of PheA* prephenate dehydratase to thermal inactivation was particularly striking.(ABSTRACT TRUNCATED AT 250 WORDS)
Project description:Buchnera aphidicola, the prokaryotic endosymbiont of aphids, complements dietary deficiencies with the synthesis and provision of several essential amino acids. We have cloned and sequenced a region of the genome of B. aphidicola isolated from Acyrthosiphon pisum which includes the two-domain aroQ/pheA gene. This gene encodes the bifunctional chorismate mutase-prephenate dehydratase protein, which plays a central role in L-phenylalanine biosynthesis. Two changes involved in the overproduction of this amino acid have been detected. First, the absence of an attenuator region suggests a constitutive expression of this gene. Second, the regulatory domain of the Buchnera prephenate dehydratase shows changes in the ESRP sequence, which is involved in the allosteric binding of phenylalanine and is strongly conserved in prephenate dehydratase proteins from practically all known organisms. These changes suggest the desensitization of the enzyme to inhibition by phenylalanine and would permit the bacterial endosymbiont to overproduce phenylalanine.
Project description:AIM:To study the effect of integration of tandem aroG-pheA genes into the tyrA locus of Corynebacterium glutamicum (C. glutamicum) on the production of L-phenylalanine. METHODS:By nitrosoguanidine mutagenesis, five p-fluorophenylalanine (FP)-resistant mutants of C.glutamicum FP were selected. The tyrA gene encoding prephenate dehydrogenase (PDH) of C.glutamicum was amplified by polymerase chain reaction (PCR) and cloned on the plasmid pPR. Kanamycin resistance gene (Km) and the P(BF) -aroG-pheA-T (GA) fragment of pGA were inserted into tyrA gene to form targeting vectors pTK and pTGAK, respectively. Then, they were transformed into C.glutamicum FP respectively by electroporation. Cultures were screened by a medium containing kanamycin and detected by PCR and phenotype analysis. The transformed strains were used for L-phenylalanine fermentation and enzyme assays. RESULTS:Engineering strains of C.glutamicum (Tyr(-)) were obtained. Compared with the original strain, the transformed strain C. glutamicum GAK was observed to have the highest elevation of L-phenylalanine production by a 1.71-fold, and 2.9-, 3.36-, and 3.0-fold in enzyme activities of chorismate mutase, prephenate dehydratase and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase, respectively. CONCLUSION:Integration of tandem aroG-pheA genes into tyrA locus of C. glutamicum chromosome can disrupt tyrA gene and increase the yield of L-phenylalanine production.
Project description:In addition to being a vital component of proteins, phenylalanine is also a precursor of numerous aromatic primary and secondary metabolites with broad physiological functions. In plants phenylalanine is synthesized predominantly via the arogenate pathway in plastids. Here, we describe the structure, molecular players and subcellular localization of a microbial-like phenylpyruvate pathway for phenylalanine biosynthesis in plants. Using a reverse genetic approach and metabolic flux analysis, we provide evidence that the cytosolic chorismate mutase is responsible for directing carbon flux towards cytosolic phenylalanine production via the phenylpyruvate pathway. We also show that an alternative transcription start site of a known plastidial enzyme produces a functional cytosolic prephenate dehydratase that catalyzes the conversion of prephenate to phenylpyruvate, the intermediate step between chorismate mutase and phenylpyruvate aminotransferase. Thus, our results complete elucidation of phenylalanine biosynthesis via phenylpyruvate in plants, showing that this pathway splits from the known plastidial arogenate pathway at chorismate, instead of prephenate as previously thought, and the complete pathway is localized in the cytosol.
Project description:Mandelic acid is an important aromatic fine chemical and is currently mainly produced via chemical synthesis. Recently, mandelic acid production was achieved by microbial fermentations using engineered Escherichia coli and Saccharomyces cerevisiae expressing heterologous hydroxymandelate synthases (hmaS). The best-performing strains carried a deletion of the gene encoding the first enzyme of the tyrosine biosynthetic pathway and therefore were auxotrophic for tyrosine. This was necessary to avoid formation of the competing intermediate hydroxyphenylpyruvate, the preferred substrate for HmaS, which would have resulted in the predominant production of hydroxymandelic acid. However, feeding tyrosine to the medium would increase fermentation costs. In order to engineer a tyrosine prototrophic mandelic acid-producing S. cerevisiae strain, we tested three strategies: (1) rational engineering of the HmaS active site for reduced binding of hydroxyphenylpyruvate, (2) compartmentalization of the mandelic acid biosynthesis pathway by relocating HmaS together with the two upstream enzymes chorismate mutase Aro7 and prephenate dehydratase Pha2 into mitochondria or peroxisomes, and (3) utilizing a feedback-resistant version of the bifunctional E. coli enzyme PheA (PheAfbr) in an aro7 deletion strain. PheA has both chorismate mutase and prephenate dehydratase activity. Whereas the enzyme engineering approaches were only successful in respect to reducing the preference of HmaS for hydroxyphenylpyruvate but not in increasing mandelic acid titers, we could show that strategies (2) and (3) significantly reduced hydroxymandelic acid production in favor of increased mandelic acid production, without causing tyrosine auxotrophy. Using the bifunctional enzyme PheAfbr turned out to be the most promising strategy, and mandelic acid production could be increased 12-fold, yielding titers up to 120?mg/L. Moreover, our results indicate that utilizing PheAfbr also shows promise for other industrial applications with S. cerevisiae that depend on a strong flux into the phenylalanine biosynthetic pathway.
Project description:Acidovorax citrulli (Ac) is a plant pathogenic bacterium that causes bacterial fruit blotch (BFB) in cucurbit crops. Despite its importance in the cucurbit industry, resistant cultivars/lines against BFB have not yet been identified. Therefore, there is a need to characterize the virulence factors/mechanisms in Ac to control the disease. Chorismate mutase, a key enzyme in the shikimate pathway, produces aromatic amino acids. Here, we report the functions of putative bifunctional chorismate mutase/prephenate dehydratase in Ac (CmpAc) determined by proteomic analysis and phenotypic assays. Ac strain lacking CmpAc, Ac?cmpAc(EV), were significantly less virulent on watermelon in the germinated-seed inoculation and leaf infiltration assays. Sequence analysis revealed that CmpAc possesses two distinct domains: chorismate mutase and prephenate dehydratase, indicating that CmpAc is a bifunctional protein. Auxotrophic assays demonstrated that CmpAc is required for the biosynthesis of phenylalanine, but not tyrosine. The comparative proteomic analysis revealed that CmpAc is mostly involved in cell wall/membrane/envelop biogenesis. Furthermore, Ac?cmpAc(EV) showed reduced twitching halo production and enhanced biofilm formation. In addition, Ac?cmpAc(EV) was less tolerant to osmotic stress but more tolerant to antibiotics (polymyxin B). Thus, our study provides new insights into the functions of a putative bifunctional protein related to virulence in Ac.
Project description:- Identification of proteins whose expression was affected by a bifunctional chorismate mutase/prephenate dehydratase in Acidovorax citrulli str. KACC17005 - Shotgun proteomic analysis was used - Two strains were used with three biological replicates (total 6 samples). WT: the wild-type strain. 12G: a bifunctional chorismate mutase/prephenate dehydratase knockout mutant
Project description:Phenylalanine (Phe) is a precursor for a large group of plant specialized metabolites, including the fragrant volatile benzenoid-phenylpropanoids (BPs). In plants, the main pathway leading to production of Phe is <i>via</i> arogenate, while the pathway <i>via</i> phenylpyruvate (PPY) is considered merely an alternative route. Unlike plants, in most microorganisms the only pathway leading to the synthesis of Phe is <i>via</i> PPY. Here we studied the effect of increased PPY production in petunia on the formation of BPs volatiles and other specialized metabolites originating from Phe both in flowers and leaves. Stimulation of the pathway <i>via</i> PPY was achieved by transforming petunia with <i>PheA<sup>?</sup></i> , a gene encoding a bacterial feedback insensitive bi-functional chorismate mutase/prephenate dehydratase enzyme. <i>PheA<sup>?</sup></i> overexpression caused dramatic increase in the levels of flower BP volatiles such as phenylacetaldehyde, benzaldehyde, benzyl acetate, vanillin, and eugenol. All three BP pathways characterized in petunia flowers were stimulated in <i>PheA<sup>?</sup></i> flowers. In contrast, <i>PheA<sup>?</sup></i> overexpression had only a minor effect on the levels of amino acids and non-volatile metabolites both in the leaves and flowers. The one exception is a dramatic increase in the level of rosmarinate, a conjugate between Phe-derived caffeate and Tyr-derived 3,4-dihydroxyphenylacetate, in PheA<sup>?</sup> leaves. <i>PheA<sup>?</sup></i> petunia flowers may serve as an excellent system for revealing the role of PPY in the production of BPs, including possible routes directly converting PPY to the fragrant volatiles. This study emphasizes the potential of the PPY route in achieving fragrance enhancement in flowering plants.
Project description:3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAHPS) catalyzes the first step in the biosynthesis of a number of aromatic metabolites. Likely because this reaction is situated at a pivotal biosynthetic gateway, several DAHPS classes distinguished by distinct mechanisms of allosteric regulation have independently evolved. One class of DAHPSs contains a regulatory domain with sequence homology to chorismate mutase-an enzyme further downstream of DAHPS that catalyzes the first committed step in tyrosine/phenylalanine biosynthesis-and is inhibited by chorismate mutase substrate (chorismate) and product (prephenate). Described in this work, structures of the Listeria monocytogenes chorismate/prephenate regulated DAHPS in complex with Mn(2+) and Mn(2+) + phosphoenolpyruvate reveal an unusual quaternary architecture: DAHPS domains assemble as a tetramer, from either side of which chorismate mutase-like (CML) regulatory domains asymmetrically emerge to form a pair of dimers. This domain organization suggests that chorismate/prephenate binding promotes a stable interaction between the discrete regulatory and catalytic domains and supports a mechanism of allosteric inhibition similar to tyrosine/phenylalanine control of a related DAHPS class. We argue that the structural similarity of chorismate mutase enzyme and CML regulatory domain provides a unique opportunity for the design of a multitarget antibacterial.
Project description:The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.
Project description:Methanococcus maripaludis is a strictly anaerobic, methane-producing archaeon. Aromatic amino acids (AroAAs) are biosynthesized in this autotroph either by the de novo pathway, with chorismate as an intermediate, or by the incorporation of exogenous aryl acids via indolepyruvate oxidoreductase (IOR). In order to evaluate the roles of these pathways, the gene that encodes the third step in the de novo pathway, 3-dehydroquinate dehydratase (DHQ), was deleted. This mutant required all three AroAAs for growth, and no DHQ activity was detectible in cell extracts, compared to 6.0 +/- 0.2 mU mg(-1) in the wild-type extract. The growth requirement for the AroAAs could be fulfilled by the corresponding aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate. The specific incorporation of phenylacetate into phenylalanine by the IOR pathway was demonstrated in vivo by labeling with [1-(13)C]phenylacetate. M. maripaludis has two IOR homologs. A deletion mutant for one of these homologs contained 76, 74, and 42% lower activity for phenylpyruvate, p-hydoxyphenylpyruvate, and indolepyruvate oxidation, respectively, than the wild type. Growth of this mutant in minimal medium was inhibited by the aryl acids, but the AroAAs partially restored growth. Genetic complementation of the IOR mutant also restored much of the wild-type phenotype. Thus, aryl acids appear to regulate the expression or activity of the de novo pathway. The aryl acids did not significantly inhibit the activity of the biosynthetic enzymes chorismate mutase, prephenate dehydratase, and prephenate dehydrogenase in cell extracts, so the inhibition of growth was probably not due to an effect on these enzymes.