Cloning, sequencing, and expression of the P-protein gene (pheA) of Pseudomonas stutzeri in Escherichia coli: implications for evolutionary relationships in phenylalanine biosynthesis

The pheA gene encoding the bifunctional P-protein (chorismate mutase:prephenate dehydratase) was cloned from Pseudomonas stutzeri and sequenced. This is the first gene of phenylalanine biosynthesis to be cloned and sequenced from Pseudomonas. The pheA gene was expressed in Escherichia coli, allowing complementation of an E. coli pheA auxotroph. The enzymic and physical properties of the P-protein from a recombinant E. coli auxotroph expressing the pheA gene were identical to those of the native enzyme from P. stutzeri. The nucleotide sequence of the P. stutzeri pheA gene was 1095 base pairs in length, predicting a 365-residue protein product with an Mr of 40,844. Codon usage in the P. stutzeri pheA gene was similar to that of Pseudomonas aeruginosa but unusual in that cytosine and guanine were used at nearly equal frequencies in the third codon position. The deduced P-protein product showed sequence homology with peptide sequences of the E. coli P-protein, the N-terminal portion of the E. coli T-protein (chorismate mutase:prephenate dehydrogenase), and the monofunctional prephenate dehydratases of Bacillus subtilis and Corynebacterium glutamicum. A narrow range of values (26-35%) for amino acid matches revealed by pairwise alignments of monofunctional and bifunctional proteins possessing activity for prephenate dehydratase suggests that extensive divergence has occurred between even the nearest phylogenetic lineages.     

Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.

The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.     

Novel mutations in the pheA gene of Escherichia coli K-12 which result in highly feedback inhibition-resistant variants of chorismate mutase/prephenate dehydratase.

The bifunctional enzyme chorismate mutase/prephenate dehydratase (EC 5.4.99.5/4.2.1.51), which is encoded by the pheA gene of Escherichia coli K-12, is subject to strong feedback inhibition by L-phenylalanine. Inhibition of the prephenate dehydratase activity is almost complete at concentrations of L-phenylalanine greater than 1 mM. The pheA gene was cloned, and the promoter region was modified to enable constitutive expression of the gene on plasmid pJN302. As a preliminary to sequence analysis, a small DNA insertion at codon 338 of the pheA gene unexpectedly resulted in a partial loss of prephenate dehydratase feedback inhibition. Four other mutations in the pheA gene were identified following nitrous acid treatment of pJN302 and selection of E. coli transformants that were resistant to the toxic phenylalanine analog beta-2-thienylalanine. Each of the four mutations was located within codons 304 to 310 of the pheA gene and generated either a substitution or an in-frame deletion. The mutations led to activation of both enzymatic activities at low phenylalanine concentrations, and three of the resulting enzyme variants displayed almost complete resistance to feedback inhibition of prephenate dehydratase by phenylalanine concentrations up to 200 mM. In all four cases the mutations mapped in a region of the enzyme that has not been implicated previously in feedback inhibition sensitivity of the enzyme.     

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.

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)     

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.

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)     

Cyclohexadienyl dehydratase from Pseudomonas aeruginosa. Molecular cloning of the gene and characterization of the gene product.

The gene encoding cyclohexadienyl dehydratase (denoted pheC) was cloned from Pseudomonas aeruginosa by functional complementation of a pheA auxotroph of Escherichia coli. The gene was highly expressed in E. coli due to the use of the high-copy number vector pUC18. The P. aeruginosa cyclohexadienyl dehydratase expressed in E. coli was purified to electrophoretic homogeneity. The latter enzyme exhibited identical physical and biochemical properties as those obtained for cyclohexadienyl dehydratase purified from P. aeruginosa. The activity ratios of prephenate dehydratase to arogenate dehydratase remained constant (about 3.3-fold) throughout purification, thus demonstrating a single protein having broad substrate specificity. The cyclohexadienyl dehydratase exhibited Km values of 0.42 mM for prephenate and 0.22 mM for L-arogenate, respectively. The pheC gene was 807 base pairs in length, encoding a protein with a calculated molecular mass of 30,480 daltons. This compares with a molecular mass value of 29.5 kDa determined for the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since the native molecular mass determined by gel filtration was 72 kDa, the enzyme probably is a homodimer. Comparison of the deduced amino acid sequence of pheC from P. aeruginosa with those of the prephenate dehydratases of Corynebacterium glutamicum, Bacillus subtilis, E. coli, and Pseudomonas stutzeri by standard pairwise alignments did not establish obvious homology. However, a more detailed analysis revealed a conserved motif (containing a threonine residue known to be essential for catalysis) that was shared by all of the dehydratase proteins.     

Regulatory gene of phenylalanine biosynthesis (pheR) in Salmonella typhimurium

4-Fluorophenylalanine-resistant mutants of Salmonella typhimurium were isolated in which synthesis of chorismate mutase P-prephenate dehydratase (specified by pheA) was highly elevated. Transduction analysis showed that the mutation affecting pheA activity was not linked to pheA, and conjugation and merodiploid analysis indicated that it was in the 95- to 100-min region of the Salmonella chromosome. Evidence is presented for the hypothesis that the mutation responsible for constitutivity of chorismate mutase P-prephenate dehydratase occurred in pheR, a gene specifying a cytoplasmic product that affected pheA. pheR mutants were found to carry a second mutation, tyrO. The tyrO mutation acts cis to cause increased levels of the tyrosine biosynthetic enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthetase (tyr) and prephenate dehydrogenase, but it has no effect on regulation of pheA.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

Prephenate dehydratase from the aphid endosymbiont (Buchnera) displays changes in the regulatory domain that suggest its desensitization to inhibition by phenylalanine

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.     

The pheR gene of Escherichia coli encodes tRNA(Phe), not a repressor protein

Nucleotide sequence analysis and transposon 5 (Tn5) insertional mutagenesis indicate that the Escherichia coli gene pheR encodes tRNA(Phe) and not a repressor protein as previously reported. The coding region of pheR is identical to that of three other cloned tRNA(Phe) genes, pheU, pheV, and pheW. Multicopy plasmids carrying pheR, like those carrying pheU, pheV, or pheW, complement a temperature-sensitive lesion in the gene for the alpha-subunit of phenylalanyl-tRNA synthetase (pheS). The nucleotide sequences of the 5'-flanking DNA of pheR, pheU, and pheW are almost identical but are quite different from the same region of pheV. By comparison with pheV, which has two tandem promoters, pheR was found to have a single promoter. The expression of pheA (encoding chorismate mutase/prephenate dehydratase) in strains carrying the pheR374 allele was decreased to similar extents by multicopy plasmids containing either pheR or pheV. It is proposed that this decrease in pheA expression and the increase in expression of pheA previously reported for chromosomal pheR mutants are both mediated through the attenuation control mechanism that regulates pheA.     

Molecular cloning of pheR in Escherichia coli K-12.

The regulator gene pheR, which in Escherichia coli controls the expression of pheA, the structural gene for chorismate mutase P-prephenate dehydratase, was cloned on to multicopy plasmids directly from the E. coli chromosome; this was achieved with the aid of the tetracycline resistance transposon, Tn10, that had been inserted very close to the pheR gene. Subsequently, pheR was subcloned on a 1.1-kilobase-pair fragment on the plasmid vector pBR322; its position on the plasmid was localized by the method of gamma delta-mediated transpositional inactivation. The pheR gene product was identified in maxicells and found to be a protein of subunit molecular weight 19,000, suggesting that the coding segment of the gene is about 500 nucleotide pairs long.     

大肠杆菌苯丙氨酸生物合成关键酶的串联表达

ａｒｏＧ基因编码的 ３－脱氧－２－阿拉伯庚酮糖－７－磷酸合成酶（ＤＡＨＰ　Ｓｙｎｔｈｅｔａｓｅ　ＤＳ）和 ｐｈｅＡ基因编码的分支酸变位酶／预苯酸脱水酶（Ｃｈｏｒｉｍａｔｅ ｍｕｔａｓｅ／ Ｐｒｅｐｈｅｎａｔｅ　ｄｅｈｙｄｒａｔａｓｅ,ＣＷ／ＰＤ）都是本丙氨酸合成途径中的关键酶,为了通过基因工程手段来增加本丙氨酸生物的产量,在利用高效的原核表达载体ｐＢＶ２２ ０对ｐｈｅＡ基因编码的ＣＭ／ ＰＤ 酶进行了表达的基础上,采用ＰＣＲ方法扩增了抗反馈抑制的ａｒｃＧ基因,进行克隆表达,并与ｐｈｅＡ基因串联,以ＰＲＰＬ－ａｒｏＧ－ＰＬ－ｐｈｅＡ的形式,实现了２种酶基因在大肠杆菌中的表达, ＳＤＳＰＡＧＥ 图谱显示了新增的４３ｋｕ及３５ｋｕ蛋白带,经酶活性测定ＤＳ、ＣＭ／ＰＤ酶的比活分别提高了 ４．６７倍、８０５／１０．７１倍。     

Chorismate mutase:prephenate dehydratase from Acinetobacter calcoaceticus. Purification, properties and immunological cross-reactivity

The bifunctional P protein (chorismate mutase: prephenate dehydratase) from Acinetobacter calcoaceticus has been purified. It was homogeneous in polyacrylamide gels and was more than 95% pure on the basis of the immunostaining of purified P protein with the antibodies raised against the P protein. The native enzyme is a homodimer (Mr = 91,000) composed of 45-kDa subunits. A twofold increase in the native molecular mass of the P protein occurred in the presence of L-phenylalanine (inhibitor of both activities) or L-tyrosine (activator of the dehydratase activity) during gel filtration. Chorismate mutase activity followed Michaelis-Menten kinetics with a Km of 0.55 mM for chorismate. L-Phenylalanine was a relatively poor non-competitive inhibitor of the mutase activity. The chorismate mutase activity was also competitively inhibited by prephenate (reaction product). Substrate-saturation curves for the dehydratase activity were sigmoidal showing positive cooperativity among the prephenate-binding sites. L-Tyrosine activated prephenate dehydratase strongly but did not abolish positive cooperativity with respect to prephenate. L-Phenylalanine inhibited the dehydratase activity, and the substrate-saturation curves became increasingly sigmoidal as phenylalanine concentrations were increased with happ values changing from 2.0 (no phenylalanine) to 4.0 (0.08 mM L-phenylalanine). A sigmoidal inhibition curve of the dehydratase activity by L-phenylalanine gave Hill plots having a slope of -2.9. Higher ionic strength increased the dehydratase activity by reducing the positive cooperative binding of prephenate, and the sigmoidal substrate-saturation curves were changed to near-hyperbolic form. The happ values decreased with increase in ionic strength. Antibodies raised against the purified P protein showed cross-reactivity with the P proteins from near phylogenetic relatives of A. calcoaceticus. At a greater phylogenetic distance, cross-reaction was superior with P protein from Neisseria gonorrhoeae than with that from the more closely related Escherichia coli.     

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.     

pheA (Rv3838c) of Mycobacterium tuberculosis encodes an allosterically regulated monofunctional prephenate dehydratase that requires both catalytic and regulatory domains for optimum activity

Prephenate dehydratase (PDT) is a key regulatory enzyme in l-phenylalanine biosynthesis. In Mycobacterium tuberculosis, expression of pheA, the gene encoding PDT, has been earlier reported to be iron-dependent (1, 2). We report that M. tuberculosis pheA is also regulated at the protein level by aromatic amino acids. All of the three aromatic amino acids (phenylalanine, tyrosine, and tryptophan) are potent allosteric activators of M. tuberculosis PDT. We also provide in vitro evidence that M. tuberculosis PDT does not possess any chorismate mutase activity, which suggests that, unlike many other enteric bacteria (where PDT exists as a fusion protein with chorismate mutase), M. tuberculosis PDT is a monofunctional and a non-fusion protein. Finally, the biochemical and biophysical properties of the catalytic and regulatory domains (ACT domain) of M. tuberculosis PDT were studied to observe that, in the absence of the ACT domain, the enzyme not only loses its regulatory activity but also its catalytic activity. These novel results provide evidence for a monofunctional prephenate dehydratase enzyme from a pathogenic bacterium that exhibits extensive allosteric activation by aromatic amino acids and is absolutely dependent upon the presence of catalytic as well as the regulatory domains for optimum enzyme activity.     

Metabolic Engineering To Produce Tyrosine or Phenylalanine in a Tryptophan-Producing Corynebacterium glutamicum Strain

The aromatic amino acids are synthesized via a common biosynthetic pathway. A tryptophan-producing mutant of Corynebacterium glutamicum was genetically engineered to produce tyrosine or phenylalanine in abundance. To achieve this, three biosynthetic genes encoding the first enzyme in the common pathway, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DS), and the branch-point enzymes chorismate mutase and prephenate dehydratase were individually cloned from regulatory mutants of C. glutamicum which have either of the corresponding enzymes desensitized to end product inhibition. These cloned genes were assembled one after another onto a multicopy vector of C. glutamicum to yield two recombinant plasmids. One plasmid, designated pKY1, contains the DS and chorismate mutase genes, and the other, designated pKF1, contains all three biosynthetic genes. The enzymes specified by both plasmids were simultaneously overexpressed approximately sevenfold relative to the chromosomally encoded enzymes in a C. glutamicum strain. When transformed with pKY1 or pKF1, tryptophan-producing C. glutamicum KY10865, with the ability to produce 18 g of tryptophan per liter, was altered to produce a large amount of tyrosine (26 g/liter) or phenylalanine (28 g/liter), respectively, because the accelerated carbon flow through the common pathway was redirected to tyrosine or phenylalanine.     

Amino acid sequences of soluble tryptic peptides of chorismate mutase/prephenate dehydratase from Escherichia coli K12.

The amino acid sequences of 28 soluble tryptic peptides from chorismate mutase/prephenate dehydratase from Escherichia coli K12 have been determined. Together with the four unique cysteine-containing peptides sequenced by Gething and Davidson ((1976) Eur. J. Biochem. 71, 327-336) this accounts for approximately 75% of the total sequence expected for this protein. A high frequency of identify between some of the peptides suggests the possibility of gene duplication during the evolution of the structural gene for the enzyme.