- Open Access
The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa
© Calhoun et al., licensee BioMed Central Ltd 2001
- Received: 2 April 2001
- Accepted: 13 June 2001
- Published: 27 July 2001
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.
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.
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.
- Periplasmic Fraction
- Chorismate Mutase
- Shikimate Dehydrogenase
- Histidine Biosynthesis
Gene families for cyclohexadienyl mutase
Assigning a name to an enzyme would be vastly more simple if all homologs belonging to a given protein family possessed identical substrate specificities. At one extreme, however, completely independent homology groups can possess the same substrate specificity; that is, a given function may have evolved more than once. At the other extreme, a single protein family may possess member enzymes that have different substrate specificities. In fact, distinct and dispersed subsets of proteins having the same substrate specificity may exist within a single protein family whose members possess an admixture of different substrate specificities. The latter case is exemplified by the α, β and γ subfamilies of aromatic aminotransferases within the aminotransferases of family I . Nevertheless, the differing substrate specificities within the catalytic repertoire of a particular protein family are generally a reflection of similar reaction mechanisms. Therefore, it should usually be feasible to name a protein family (or superfamily) at a hierarchical level that encompasses all substrate specificities of its member proteins. Chorismate mutase, isochorismate mutase  and 4-amino, 4-deoxychorismate mutase , which all catalyze similar reactions, are common members of a group that we have called cyclohexadienyl mutase (Table 1). In this vein, class I cyclohexadienyl mutases possess chorismate mutase members that belong to either family Iα (AroQ) or family Iβ (AroR). A third group of chorismate mutases (AroH) so far constitute the sole members of a separate homology group, the class II cyclohexadienyl mutases.
Chorismate mutases encoded by both aroQ and aroH are represented among prokaryotes. The distribution of AroH is quite limited, known at this time to be present only in some Bacillus species (B. subtilis, B. halodurans, and B. stearothermophilus), cyanobacteria (Synechocystis sp., Synechococcus sp., Nostoc punctiforme, Anabaena sp., and Prochlorococcus marinus), Streptomyces coelicolor, and Thermomonospora fusca. On the other hand, AroQ is widely distributed. Multiple paralogs of aroQ are commonly present in a single organism. Genes encoding AroQ and AroH coexist in some representatives of Bacillus. The AroQ proteins are all-helix-bundle domains , whereas the AroH proteins possess a trimeric pseudo α/β-barrel structure . The AroR proteins (known only in eukaryotes) are structurally similar to AroQ, deploying a four-helix-bundle structure around the active site. The AroR and AroQ proteins also share a colinear arrangement of amphiphilic patterns, as well as conserved spacing between patches of closely neighboring patterns in the primary sequence. Thus, AroR can be viewed as a version of AroQ that has inserted loops related to allosteric regulation. The latter rationale is the basis for classification of AroQ and AroR into the same homology group ( and references therein) even though homology is barely detectable at the sequence level. Both AroQ and AroH are small (20 kDa) proteins. AroR is much larger and exhibits complex allostery, being activated by L-tryptophan and inhibited by L-tyrosine in the case of yeast  or being activated by L-tryptophan and inhibited by both L-tyrosine and L-phenylalanine in the case of higher plants .
Nature of gene product
•aroQ or aroQ•
Fused with another domain
AroQ p †
AroQ t †
Among the monofunctional chorismate mutases, that present in Erwinia herbicola possesses a cleavable signal peptide and accordingly is translocated into the periplasm. E. herbicola chorismate mutase was shown  to be not only periplasmic but also located at the poles of the cells, similar to cyclohexadienyl dehydratase of Pseudomonas aeruginosa . In this study we have examined the extent to which monofunctional chorismate mutases are periplasmic or cytoplasmic. A chorismate mutase of Salmonella typhimurium is shown to be periplasmic. A periplasmic chorismate mutase in P. aeruginosa is shown to be part of an complete three-step pathway for periplasmic conversion of chorismate to phenylalanine.
Chorismate mutase is the focal point of this paper, and Figure 1 illustrates that the cytoplasm of S. typhimurium has two paralogs of the Iα family of chorismate mutases denoted AroQ. One is fused to TyrA and the other to PheA, as indicated by the peg-and-hole depiction in the figure. A third AroQ paralog of the Iα group is present in the periplasm (*AroQ). P. aeruginosa is similar to S. typhimurium in having an AroQ•PheA fusion in the cytoplasm as well as the periplasmic AroQ species (*AroQ), but differs in lacking the AroQ•TyrA fusion. Multidomain proteins whose domains have separable counterparts elsewhere must be appropriately named to facilitate comparisons (see Table 2).
Chorismate-to-phenylalanine pathway in the P. aeruginosaperiplasm
A modified magnesium shock procedure was used to separate periplasmic and cytoplasmic fractions of P. aeruginosa cultures grown in minimal medium. The established periplasmic enzyme *PheC [13,16] was used as a periplasmic protein marker and shikimate dehydrogenase was used as a cytoplasmic protein marker. It was preferable to use prephenate rather than L-arogenate as a substrate in the assay of *PheC activity, not only because the assay for L-arogenate dehydratase is tedious  but also because prephenate is much less expensive. However, *PheC cannot be monitored as prephenate dehydratase in the presence of cytoplasmic P-protein because the PheA domain of that protein is prephenate dehydratase. To avoid this problem, the aroQ•pheA-deficient mutant PAT1051 was used. All the prephenate dehydratase activity in PAT1051 is derived from *PheC, which thus can be used as a convenient marker for the quality of the separation of the cellular compartments.
The presence of chorismate mutase and aromatic aminotransferase in the periplasmic fraction of Pseudomonas aeruginosa
Total enzyme activities†
*AroQ + AroQ•
*AroQ was found in the periplasm in P. aeruginosa PAO1. None of the periplasmic chorismate mutase activity was attributed to AroQ•PheA because no PheA-domain activity was detected (Table 3). Hence, the chorismate mutase activity in the periplasm must be exclusively *AroQ. Identification of this activity as *AroQ, and not AroQ•PheA, was verified by elution of a single peak of chorismate mutase in the wash fraction recovered after Mono Q chromatography of the periplasmic fraction. This is the established elution behavior of *AroQ . Of the total aromatic aminotransferase activity, 10–20% was found in the periplasm.
Purification of *AroQ and *AatIβ from P. aeruginosaperiplasm
Purification of *AroQ from Pseudomonas aeruginosa
Mono Q (pH 7.0)
Mono Q (pH 8.4)
Aromatic aminotransferase (*AatIβ) was purified from the periplasmic fraction of PAO1. The protein fractions with enzymatic activity that eluted from the gradient of a hydroxylapatite column were chromatographed on a mono Q column and recovered as single peaks. The leading peak fractions yielded one major band and several minor bands after SDS-PAGE. Amino-terminal amino-acid sequencing of the major band gave GSGATSAAGK, which corresponds to the sequence beginning with residue 29 of the deduced protein encoded by PA2531 in the completed P. aeruginosa genome . In addition, one of the minor bands exhibited the sequence AGKPQETFLD, which corresponds to the sequence of the protein encoded by gene PA2531 beginning at residue 36. Thus, a favored and a minor cleavage site seem to exist for the signal peptide. The Mr estimated by SDS-PAGE staining with Coomassie blue was about 37,000. The Mr estimated by gel filtration was 73,000, indicating the presence of a homodimer.
Isolation of S. typhimurium*AroQ
E. coli BL21(pJB-1) containing *aroQ from S. typhimurium was grown to late-exponential growth in 1 liter of LB medium containing kanamycin. After cell harvest by centrifugation, 1.5 g of wet cells were subjected to osmotic shock . After centrifugation, the supernatant was collected and designated as the shocked periplasmic fraction. An equal portion of wet cells, treated identically but not subjected to osmotic shock, was designated as the non-shock periplasmic fraction. Crude extracts were also prepared from each cell-pellet fraction.
The shocked periplasmic fraction contained 26,100 units (1 unit = 1 nmol of prephenate produced per minute) of chorismate mutase activity. As the prephenate dehydratase and prephenate dehydrogenase activities associated with the cytoplasmic AroQ•PheA and AroQ•TyrA proteins were not detected in this fraction, contamination with cytoplasmic species of AroQ was less than 1%. The additional assay for shikimate dehydrogenase as a marker for cytoplasmic contamination also showed less than 1% of total activity to be in the shocked periplasmic fraction. On the other hand, 97% of the total alkaline phosphatase (periplasmic marker) was recovered in the shocked periplasmic fraction. Although cross-reactivity was relatively poor, the presence of S. typhimurium *AroQ in periplasmic fractions and its absence in cytoplasmic fractions was demonstrated by western blotting using antibody obtained against E. herbicola AroQ .
Enzymological characterization of periplasmic chorismate mutases
Kinetic constants, pI and Mw of *AroQ enzymes
8.77 / 20,417
8.04 / 17,939
7.02 / 20,646
6.58 / 18,392
6.28 / 20,299
6.24 / 18,017
6.43 / 20,761
5.66 / 17,504
5.40 / 21,945
5.03 / 18,475
6.58 / 22,087
5.90 / 19,460
New periplasmic subgroup of AroQ
When the periplasmic chorismate mutase from E. herbicola was initially cloned and sequenced, it was a unique case of an AroQ protein having a cleavable signal peptide and a carboxy-terminal extension. The current repertoire of additional AroQ proteins that can be gleaned from the contemporary databases on the criterion of these two features numbers eight. One of them has been located in the esophageal glands of the phytoparasitic nematode Meloidogyne javanica. Presumably, this AroQ protein is secreted into plant cells as part of the parasitic process . The erratic phylogenetic distribution of *AroQ may have a variety of explanations that include massive gene loss, horizontal transfer or independent evolution of a signal peptide and carboxy-terminal extension.
Figure 2 shows a multiple sequence alignment which highlights conserved amino-acid residues in the *AroQ subfamily in relationship to the homologous residues that have been shown by X-ray crystallography  to be located in the active site of the AroQ domain of E. coli AroQ•PheA. *AroQ proteins are twice the size of the basic AroQ catalytic domain. The carboxy-terminal half of each *AroQ protein is of similar length but not highly conserved, and it has no known catalytic or regulatory function. When used as a query sequence, it returns no significant hits. One reasonable speculation is that as *AroQ maybe localized at the polar cap(s) of the cell, this region might accomplish some sort of protein-protein or protein-membrane interaction that might facilitate its spatial location at the polar regions of the periplasmic milieu.
Substrate specificities of AroQ proteins
Chorismate mutase catalyzes a facile rearrangement reaction that can progress to a significant extent under nonenzymatic conditions. The aroQ genes are easily missed during genome annotation because they are small and do not show strong sequence conservation. We have identified many initially dubious AroQ hits to BLAST queries by alignment of amino-acid residues known to be critical for catalysis.
There is precedent for the finding that within a homolog grouping whose members have differing substrate specificities, proteins sharing a substrate specificity do not necessarily cluster together; for example, B. subtilis AroA and close relatives are clustered more specifically with KdsA sequences than with E. coli AroA and its close relatives . The phylogram tree at the left in Figure 3 illustrates this for the AroQ protein family. The current limitations of homology detection are such that the monofunctional AroQ proteins from Aeropyrum pernix, Enterococcus faecalis, Helicobacter pylori and Methanococcus jannaschii cannot be shown to be closer to one another or to any other AroQ proteins/domains than they are to PapB or PchB. This is because bootstrap values only support the branching nodes to the level shown in bold. Thus, even though the computational program used placed A. pernix AroQ with the valid grouping of Streptomyces coelicolor AroQ, C. difficile AroQ, and Mycobacterium tuberculosis AroQ, the A. pernix AroQ sequence could in fact be closer to any other group. Thus, one can recognize distinct and valid groupings (for example, the *AroQ periplasmic chorismate mutases), but not necessarily the exact relationship of these groupings with one another.
The AroQ family of chorismate mutases
In the five-member group labeled PchB in Figure 4, the P. aeruginosa protein has been shown to participate in salicylate synthesis and the proposed reaction (catalyzed by isochorismate mutase) has been discussed in the previous section. The P. fluorescens protein undoubtedly has the same role. This substrate specificity does not necessarily apply to the remaining members. In P. aeruginosa, pchA (isochorismate synthase) and pchB are translationally coupled, and cis-expression is obligatory for activity . In Vibrio vulnificus, pchB is closely linked to venB (isochorismatase) . V. cholerae does not exhibit a pchB homolog upstream of the gene encoding isochorismatase (vibF) . Perhaps the clustering of PchB proteins together reflects their interaction with proteins able to bind isochorismate.
The periplasmic pathway of phenylalanine biosynthesis
It has been known for some time that phenylalanine-pathway redundancy exists in P. aeruginosa. The enzymological basis for this became apparent with the recognition of the presence of an 'extra' species of chorismate mutase (CM-F) and cyclohexadienyl dehydratase in addition to the E. coli-like enzymes . This redundancy explained the "reluctant auxotrophy" of P. aeruginosa , as well as the "hidden overflow pathway" to phenylalanine . The latter phenomenon is one whereby early-pathway regulatory mutations that increase intracellular chorismate concentrations result in exclusive metabolite flow toward L-phenylalanine because cyclohexadienyl dehydratase is insensitive to allosteric control. In the periplasmic system a mixture of flow routes (chorismate → prephenate → phenylpyruvate → phenylalanine or chorismate → prephenate → arogenate → phenylalanine) exist because of the ability of cyclohexadienyl dehydratase to accept either prephenate or arogenate and because of the ability of periplasmic aminotransferase (whose detailed characterization is not within the scope of this paper) to transaminate either phenylpyruvate or prephenate.
The spatial location of the overflow pathway in the periplasmic compartment was first indicated by the demonstration that cyclohexadienyl dehydratase had a cleavable amino terminus and was translocated to the periplasm. Indirect evidence favoring a periplasmic location for chorismate mutase as well in P. aeruginosa was the demonstration of a periplasmic chorismate mutase in E. herbicola . The possible periplasmic location of an aromatic aminotransferase has been anticipated, but not directly examined until now. This paper demonstrates the identity and presence of all three enzymes constituting the overflow pathway in a single organism.
On the basis of broad comparative enzymological surveys [18,29], a similar intact overflow pathway can be anticipated to be present in Serratia and Erwinia. The application of queries to the finished and unfinished genomes at the National Center for Biotechnological Information  returned hits for periplasmic cyclohexadienyl dehydratase and periplasmic chorismate mutase in Yersinia pestis. Although no aminotransferase gene has yet been found in Y. pestis that has a predicted signal peptide, one is present that is predicted by P-Sort to be an inner-membrane protein.
Periplasmic pathway remnants?
Before the recognition of their periplasmic location, it was known  that some organisms might have *AroQ but not *PheC (Salmonella) or *PheC but not *AroQ (Klebsiella). It was pointed out that appropriate mutants are better producers of prephenate in Salmonella than in Escherichia because of the presence of *AroQ in the former but not in the latter. Detailed comparative enzymological work in enteric bacteria [18,29,32] indicates that many additional *aroQ genes will be identified. Within Enterocluster-1, Enterobacter cloacae resembles S. typhimurium in lacking *pheC but having *aroQ, whereas Enterobacter aerogenes resembles K. pneumoniae in its possession of *pheC but its absence of *aroQ. Other Enterocluster-1 members (Escherichia, Shigella, Enterobacter agglomerans (ATCC 29915), and Citrobacter,) lack both periplasmic enzymes. On the other hand, all members of Enterocluster-2 possess both *aroQ and *pheC, except for Erwinia carotovora which lacks *pheC. These include Serratia marcescens, Serratia rubidae, Enterobacter agglomerans (ATCC 27155), Erwinia herbicola, E. milletiae, E. ananas, E. tracheiphila, E. chrysanthemi and E. amylovora. Members of Enterocluster-3 (species of Cedecea, Kluyvera, Edwardsiella, Hafnia, Yersinia, Proteus, Providencia and Morganella) lack both *aroQ and *pheC.
All four species of Mycobacterium in the finished genomes resemble Salmonella in having *aroQ but lacking *pheC. The physiological function of a partial pathway is unknown; perhaps *AroQ interfaces with a different pathway. In fact, it must be conceded that even the function of the intact periplasmic pathway to phenylalanine is unknown. An important clue presumably is the spatial location at the pole(s) of the cell. The polar, periplasmic location of a number of protein systems is receiving increasing attention .
Most aminotransferases that are capable of functioning as aromatic aminotransferases are members of family I within the aminotransferase protein superfamily . These can usually be assigned to one of three subfamilies, Subfamily Iα partitions into narrow-specificity aspartate aminotransferases and broad-specificity aspartate aminotransferases that can accommodate the aromatic ring. Likewise, subfamily Iβ partitions into narrow-specificity imidazole acetol phosphate (IAP) aminotransferases and broad-specificity IAP aminotransferases that utilize aromatic amino/keto acids. Subfamily Iγ partitions into subgroups having a variety of substrate specificities, with aspartate aminotransferases being the most prominent subcluster. Among these are broad-specificity aspartate aminotransferases with ability to function with aromatic amino acids.
The periplasmic aminotransferase of P. aeruginosa belongs to the Iβ subfamily and exhibits catalytic activity as IAP aminotransferase in vitro assayed as described by Gu et al. . It exhibits signature motifs corresponding to the regions of the active-site lysine and the interdomain interface . Like its close relative, HisH of Bacillus subtilis, it has the potential to function in both histidine and aromatic amino-acid biosynthesis. Of course, the spatial location of *AatIβ in the periplasm would seem to rule out in vivo function in histidine biosynthesis. This expectation is accommodated by the observation that P. aeruginosa possesses a paralog encoded by gene PA4447  that must function in vivo as IAP aminotransferase as it is closely linked with other genes of histidine biosynthesis. Yet another paralog is encoded by gene PA3165 and carries out aromatic amino-acid biosynthesis in the cytoplasm. Thus, three subfamily Iβ paralogs of aminotransferase are expressed in P. aeruginosa: one for aromatic biosynthesis in the cytoplasm, one for histidine biosynthesis in the cytoplasm, and one for phenylalanine synthesis in the periplasm. Previous enzymological work had demonstrated the existence of five aminotransferases in P. aeruginosa that were capable of functioning as aromatic aminotransferases in vitro .
Bacterial strains, plasmids, and media
Bacterial strains and plasmids
Strain or plasmid
Genotype or description
Source or reference
F-ompT hsdSB (rB- mB-) gal dcm; with DE3, a λ prophage carrying the T7 RNA polymerase gene
BL21(DE3) transformed with pJB-1
F-φ 80dlacZΔM15 Δ (lacZYA-argF) U169 deoR recA1 endA1 up44gλ-hsdR17 (rK-, mk+) thi-1 gyrA96 relA1
62–1 (ATCC 25306)
tyrA pheA trpD
aroQpheA (leaky phenylalanine auxotroph)
T7 lac promoter, lacI+ Kmr HisTag sequence, T7Tag (11aa) sequence
pET-24b(+) containing S. typhimurium*aroQ insert
Key to sequence identifiers
E. coli transformants or P. aeruginosa cells were grown at 37°C with vigorous shaking in LB or minimal medium supplemented with appropriate antibiotics. Cells were harvested in the late-exponential phase of growth by centrifugation and resuspended in 50 mM potassium phosphate buffer (pH 7.0), containing 1 mM dithiothreitol (DTT). The cells were disrupted by sonication using an Ultratip Labsonic System (Lab-Line Instruments), and then centrifuged at 150,000 × g for 65 min at 4°C. The supernatant fraction was collected and passed through a 1.5 × 5.5 cm DG-10 Sephadex column (Bio-Rad). The resulting desalted preparation is designated as crude extract.
Pellets from BL21(DE3), the BL21(pJB-1) clone, and S. typhimurium were suspended into 25 mM potassium phosphate buffer at pH 7.0 containing 1.0 mM DTT. Extracts were sonicated using three pulses to maintain cooling. The extract was ultracentrifuged (Beckman) at 150,000 × g and 4°C. Crude extracts to be used for enzyme assays were desalted on DG-10 columns (Bio-Rad).
For routine assays with unpurified protein preparations, chorismate mutase and prephenate dehydratase were assayed spectrophotometrically by the method of Cotton and Gibson  as modified by Ahmad and Jensen , using 1 mM chorismate and 1 mM prephenate, respectively. Arogenate dehydratase was assayed by measuring the formation of L-phenylalanine using high-performance liquid chromatography (HPLC) . Aromatic aminotransferase was assayed as described before , and phenylalanine and α-ketoglutarate were used as the substrates, unless indicated otherwise. Shikimate dehydrogenase was assayed by monitoring the reduction of NADP+ at 340 nm . Alkaline phosphatase was assayed as detailed by Brickman and Beckwith .
For kinetic determinations made with purified preparations of chorismate mutase, initial rates were followed as disappearance of chorismate. This was monitored spectrophotometrically at 274 nm using an extinction coefficient of 2,630 M/cm. Steady-state parameters of Km and kcat were calculated from initial rates using a series of substrate concentrations over a 15-fold range below and above Km. Protein concentrations were estimated by the Bradford assay  with bovine serum albumin as the protein standard.
Separation of periplasmic and cytoplasmic fractions of P. aeruginosa
A modified magnesium shock procedure  was used to fractionate cytoplasmic and periplasmic enzymes in P. aeruginosa. Cultures were grown to late-exponential phase in 300 ml of M-9 salts/glucose medium. The culture was divided into two 150-ml parts and harvested by centrifugation at 6,000 × g at 4°C for 20 min. One part was resuspended in 5 ml of 50 mM potassium phosphate buffer (pH 7.0), and broken by sonication to make crude extract. The other part was resuspended in 20 ml of concentrated magnesium solution (0.2 M MgCl2, 0.01 M Tris-HCl pH 8.4), incubated on ice for 20 min, then centrifuged at 6,000 × g at 4°C for 20 min. The resulting supernatant was dialyzed against 50 mM (pH 7.0) potassium phosphate buffer overnight (using at least ten times the volume of the buffer, with one change of the buffer after about 5–6 h), concentrated on a YM-10 membrane apparatus to 4 ml, and denoted as the periplasmic fraction. The remaining pellet was resuspended in 3 ml of 50 mM phosphate buffer (pH 7.0) and sonicated to yield the cytoplasmic fraction.
*PheC  activity (assayed as prephenate dehydratase) was the marker protein for the periplasmic fraction , and shikimate dehydrogenase activity was the marker protein for the cytoplasmic fraction.
Purification of *AroQ and a periplasmic aromatic aminotransferase from P. aeruginosa
Twenty-liter cultures of P. aeruginosa strain PAO1 (1 l per 2.8–1 flask) were grown to late-exponential phase in M-9 salts/glucose medium, and the periplasmic fraction was separated from the rest of the cells as described above, but with a scaled-up protocol. All the buffers contained 1 mM DTT.
A 2.5 × 16 cm Bio-Gel HTP column previously equilibrated with 5 mM phosphate buffer (pH 7.0) containing 1 mM DTT was loaded with a 240-mg amount of protein, washed with 500 ml of starting buffer, and bound proteins were eluted with 800 ml of phosphate buffer at pH 7.0 (potassium phosphate gradient from 5–400 mM). Fractions of 3.5 ml were collected, and chorismate mutase and aromatic aminotransferase activities were assayed.
Fractions with high chorismate mutase activity were pooled and concentrated by means of an Amicon YM-10 membrane. The concentrated preparation was dialyzed overnight against 50 mM phosphate buffer (pH 7.0) containing 1 mM DTT. An FPLC-connected Mono Q HR 5/5 anion-exchange column (Pharmacia Biotech) equilibrated with 50 mM phosphate buffer (pH 7.0) containing 1 mM DTT was loaded with the concentrated *AroQ fraction. The column was washed with 10 ml of the starting buffer, and bound proteins were gradient-eluted with 20 ml of phosphate buffer (KCl gradient between 0 to 1 M). Fractions of 0.5 ml were collected, and those showing high chorismate mutase activity were pooled and concentrated by means of an Amicon YM-10 membrane. The concentrated preparation was dialyzed against 50 mM Tris-HCl buffer (pH 8.4) containing 1 mM DTT overnight, loaded onto the same Mono Q column that had been equilibrated with the same Tris-HCl buffer. The same elution regimen was used, except that the KCl gradient was from 0 to 350 mM. Fractions showing high chorismate mutase activity were pooled and dialyzed overnight against the above phosphate buffer containing 1 mM DTT and 150 mM NaCl. The fractions were concentrated by an Ultrafree-15 centrifugal filter device (Millipore Corporation), and loaded onto an FPLC-connected Superdex 75 HR 10/30 (Pharmacia Biotech) column which had been equilibrated with 50 mM phosphate buffer (pH 7.0) containing 1 mM DTT and 150 mM NaCl. The same buffer was used for protein elution and fractions of 0.5 ml were collected. The fractions showing high chorismate mutase activity were pooled and concentrated by centrifugal filtration.
Two peaks with aromatic aminotransferase activity eluted from the hydroxylapatite column mentioned above. When the major fractions with highest levels of activity were pooled and subjected to Mono Q chromatography as described above, two peaks of activity were again resolved, one in the wash fraction (minor) and one in the gradient fraction (major). This heterogeneity might be explained by the apparent existence of a second, minor, cleavage site for the signal peptide (see Results). The major peak of aromatic aminotransferase was pooled and subjected to Superdex gel filtration as described above. The fractions showing high aromatic aminotransferase activity were pooled and concentrated by centrifugal filtration.
Molecular mass determination
The molecular mass of the native enzyme was determined by gel filtration using an FPLC-connected Superdex 75 HR 10/30 column (Pharmacia Biotech). The column was operated as described above using a flow rate of 0.5 ml/min. Calibration standards were cytochrome c (Mr 12,400), carbonic anhydrase (Mr 31,000), bovine serum albumin (Mr 66,000), and alcohol dehydrogenase (Mr 150,000). The void volume was determined by blue dextran (Mr 2,000,000). SDS-PAGE was carried out to determine the subunit molecular mass of the enzyme. Lysozyme (Mr 14,400), soybean trypsin inhibitor (Mr 21,500), carbonic anhydrase (Mr 31,000), ovalbumin (Mr 45,000), bovine serum albumin (Mr 66,000) and phosphorylase b (Mr 97,400) were used as molecular mass standards.
Biochemicals and chemicals
Ampicillin, kanamycin, α-ketoglutarate, shikimate, and amino acids were purchased from Sigma Chemical Company, DTT was from Research Organics, DEAE-cellulose was from Whatman, hydroxylapatite was from Bio-Rad. Molecular mass standards for gel filtration and for SDS-PAGE were from Sigma and Bio-Rad, respectively. Growth medium components and agar were from Difco. Chorismate, prephenate, and L-arogenate are routinely prepared in this laboratory as the accumulated products of mutant microbial strains.
Cloning of the Salmonella typhimurium *aroQ gene and expression in E. coli
Standard molecular genetics techniques were used . The *aroQ gene was amplified by PCR using genomic DNA from S. typhimurium as template. The forward primer (5'-GTTATTCATATGATTCGTCATATCGCCATT-3') was designed to introduce an NdeI restriction site (CATATG) overlapping the start codon. The reverse primer (5'-GATAAACTCGAGT-TAACGCGCGTGTCGCACCA-3') was designed to introduce an XhoI site (CTCGAG) downstream of the stop codon. The PCR products were gel-purified using low melting temperature agarose and subjected to restriction enzyme digestion with NdeI and XhoI. The same two restriction enzymes were used to digest plasmid pET-24b+ DNA. The PCR fragments were ligated to the plasmid DNA and the ligation products were used to transform strain DH5α. Plasmid DNA from several kanamycin resistant colonies was used for DNA sequence analysis. A clone designated pJB-1 with the correct nucleotide sequence was used to transform strain BL21(DE3) for analysis of *aroQ expression. A well-isolated single colony was inoculated into 10 ml of LB/kanamycin medium and grown overnight at 30°C and 250 rpm. This culture was used to inoculate 100 ml of LB/kanamycin medium the following morning. Iso-propyl-thiogalactoside (IPTG) was added when cultures reached a density of about 0.6 A600, the cultures were harvested by centrifugation about 2.5 h later, and the pellets were used immediately to prepare extracts for enzyme assay.
GenBank accession numbers for the S. typhimurium *aroQ and the P. aeruginosa *aroQ and *aat sequences are AY039113, AF389865 and AF389866, respectively.
Analysis by SDS-PAGE and western blot
SDS-PAGE was performed with a Mini-PROTEIN II cell (Bio-Rad) using the method of Laemmli . Samples from pellets of centrifuged cells were used to make crude extracts or were treated for whole-cell lysis. Crude extracts were usually diluted 1:1 (v/v) with solubilization buffer and heated to 100°C for 10 min. Whole-cell lysis was performed by resuspending the pellet in 2 × SDS sample buffer, vortexing vigorously to shear the chromosomal DNA, and heating to 70°C for about 5 min. Samples from 5 to 10 μl were loaded onto SDS-12% polyacrylamide gels. MultiMark (Novex) multicolored standards were used as protein markers in order to estimate the efficiency of protein transfer and to determine the molecular mass of proteins of interest.
A Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad), which transfers in a horizontal apparatus with a platinum-coated titanium plate as anode and a stainless steel plate as cathode, was used to transfer the protein from the mini-gels to nitrocellulose membranes sandwiched between Extra Thick Blot Absorbent Filter Paper (Bio-Rad) in about 15 to 30 min.
Antigens were detected with a chemiluminescence detection system (femtoLUCENT, GENO TECH). Membranes were blocked in 5% BLOT-QuickBlocker PBST buffer (plus 0.1% Tween-20), washed three times with PBST buffer (plus 0.1% Tween-20), incubated in primary antibody diluted in femto/TBST buffer in a tray for 90 min at room temperature (or overnight at 4°C), washed five times (as above), incubated in secondary antibody-HRP (horseradish peroxidase)-conjugate (goat anti-rabbit) as above, washed four times with femto/TBST buffer followed by transfer to detection buffer and detection reagent. The blotted membranes were placed protein sides up into the film cassette with the film (Kodak X-ray, full speed blue) on top and exposed for 10 to 20 sec.
This work was supported in part by NIH grant RCMI RR08168.
- Bentley R: The shikimate pathway: a metabolic tree with many branches. Crit Rev Biochem Mol Biol. 1990, 25: 307-384.PubMedView ArticleGoogle Scholar
- Galperin MY, Walker DR, Koonin EV: Analogous enzymes: independent inventions in enzyme evolution. Genome Res. 1998, 8: 779-790.PubMedGoogle Scholar
- Jensen RA, Gu W: Evolutionary recruitment of biochemically specialized subdivisions of Family-I within the protein superfamily of aminotransferases. J Bacteriol. 1996, 178: 2161-2171.PubMedPubMed CentralGoogle Scholar
- Zamir LO, Nikolakakis A, Bonner CA, Jensen RA: Evidence for enzymatic formation of isoprephenate from isochorismate. Bioorg Med Chem Lett. 1993, 7: 1441-1446. 10.1016/S0960-894X(01)80426-1.View ArticleGoogle Scholar
- Blanc V, Gil P, Bamas-Jacques N, Lorenzon S, Zagorec M, Schleuniger J: Identification and analysis of genes from Streptomyces pristinaespiralis encoding enzymes involved in the biosynthesis of the 4-dimethylamino-L-phenylalanine precursor of pristinamycin.I. Mol Microbiol. 1997, 23: 191-202. 10.1046/j.1365-2958.1997.2031574.x.PubMedView ArticleGoogle Scholar
- Lee AY, Karplus PA, Ganem B, Clardy J: Atomic structure of the buried catalytic pocket of Escherichia coli chorismate mutase. J Am Chem Soc. 1995, 117: 3627-3628.View ArticleGoogle Scholar
- Chook YM, Ke H, Lipscomb WN: Crystal structure of the monofunctional chorismate mutase from Bacillus subtilis and its complex with a transition state analog. Proc Natl Acad Sci USA. 1993, 90: 8600-8603.PubMedPubMed CentralView ArticleGoogle Scholar
- MacBeath G, Kast P, Hilvert D: A small, thermostable, and monofunctional chorismate mutase from the archeon Methanococcus jannaschii. Biochemistry. 1998, 37: 10062-10073. 10.1021/bi980449t.PubMedView ArticleGoogle Scholar
- Sträter N, Hakansson K, Schnappauf G, Braus G, Lipsocomb WN: Crystal structure of the T state of allosteric yeast chorismate mutase and comparison with the R state. Proc Natl Acad Sci USA. 1996, 93: 3330-3334. 10.1073/pnas.93.8.3330.PubMedPubMed CentralView ArticleGoogle Scholar
- Goers SK, Jensen RA: The differential allosteric regulation of two chorismate-mutase isoenzymes of Nicotiana silvestris. Planta. 1984, 162: 117-124.PubMedView ArticleGoogle Scholar
- Jensen R, Nester EW: The regulatory significance of intermediary metabolites: control of aromatic acid biosynthesis by feedback inhibition in Bacillus subtilis. J Mol Biol. 1965, 12: 468-481.PubMedView ArticleGoogle Scholar
- Xia T, Song J, Zhao G, Aldrich H, Jensen RA: The aroQ encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola. J Bacteriol. 1993, 175: 4729-4737.PubMedPubMed CentralGoogle Scholar
- Zhao G, Xia Aldrich TH, Jensen RA: Cyclohexadienyl dehydratase from Pseudomonas aeruginosa is a periplasmic protein. J Gen Microbiol. 1993, 139: 807-813.PubMedView ArticleGoogle Scholar
- Gosset G, Bonner CA, Jensen RA: Microbial origin of "plant-type" 2-keto-3-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate/tryptophan-regulated enzyme from Xanthomonas campestris. J Bacteriol. 2001, 183: 4061-4070. 10.1128/JB.183.13.4061-4070.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Daugherty M, Vonstein V, Overbeek R, Osterman A: Archaeal shikimate kinase, a new member of the GHMP-kinase family. J Bacteriol. 2001, 183: 292-300. 10.1128/JB.183.1.292-300.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Zhao G, Xia T, Fischer RS, Jensen RA: Cyclohexadienyl dehydratase from Pseudomonas aeruginosa: molecular cloning of the gene and characterization of the gene product. J Biol Chem. 1992, 267: 2487-2493.PubMedGoogle Scholar
- Fischer R, Jensen RA: Arogenate dehydratase. Methods Enzymol. 1987, 142: 503-507.PubMedView ArticleGoogle Scholar
- Ahmad S, Jensen RA: Phylogenetic distribution of components of the overflow pathway to L-phenylalanine within the enteric lineage of bacteria. Curr Microbiol. 1988, 16: 295-302.View ArticleGoogle Scholar
- Pseudomonas Genome Project. [http://www.pseudomonas.com]
- Neu HC, Heppel LA: The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem. 1965, 40: 3685-3692.Google Scholar
- Lambert KN, Allen KD, Sussex IM: Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica. Mol Plant Microbe Interact. 1999, 12: 328-336.PubMedView ArticleGoogle Scholar
- Xie G, Brettin TS, Bonner CA, Jensen RA: Mixed-function supraoperons that exhibit overall conservation, albeit shuffled gene organization, across wide intergenomic distances within eubacteria. Microb Comp Genomics. 1999, 4: 5-28.PubMedView ArticleGoogle Scholar
- Serino L, Reimann C, Baur H, Beyeler M, Visca P, Haas D: Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa. Mol Gen Genet. 1995, 249: 217-228.PubMedView ArticleGoogle Scholar
- Subramaniam PS, Xie G, Xia T, Jensen RA: Substrate ambiguity of 3-deoxy-D -manno-octulosonate 8-phosphate synthase from Neisseria gonorrhoeae in the context of its membership in a protein family containing a subset of 3-deoxy-D- arabino-heptulosonate 7-phosphate synthases. J Bacteriol. 1998, 180: 119-127.PubMedPubMed CentralGoogle Scholar
- Litwin CM, Rayback TW, Skinner J: Role of catechol siderophore synthesis in Vibrio vulnificus virulence. Infect Immun. 1996, 64: 2834-2838.PubMedPubMed CentralGoogle Scholar
- Wyckoff EE, Stoebner JA, Reed KE, Payne SM: Cloning of a Vibrio cholerae vibriobactin gene cluster: Identification of genes required for early steps in siderophore biosynthesis. J Bacteriol. 1997, 179: 7055-7062.PubMedPubMed CentralGoogle Scholar
- Patel N, Stenmark-Cox S, Jensen RA: Enzymological basis of reluctant auxotrophy for phenylalanine and tyrosine in Pseudomonas aeruginosa. J Biol Chem. 1978, 253: 2972-2978.PubMedGoogle Scholar
- Fiske MJ, Whitaker RJ, Jensen RA: Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa. J Bacteriol. 1983, 154: 623-631.PubMedPubMed CentralGoogle Scholar
- Ahmad S, Weisburg WG, Jensen RA: Evolution of aromatic amino acid biosynthesis and application to the fine-tuned phylogenetic positioning of enteric bacteria. J Bacteriol. 1990, 172: 1051-1061.PubMedPubMed CentralGoogle Scholar
- National Center for Biotechnology Information. [http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/]
- Bonner CA, Fischer RS, Ahmad S, Jensen RA: Remnants of an ancient pathway to L-phenylalanine and L-tyrosine in enteric bacteria: evolutionary implications and biotechnological impact. Appl Environ Microbiol. 1990, 56: 3741-3747.PubMedPubMed CentralGoogle Scholar
- Xia T, Jensen RA: Monofunctional chorismate mutase from Serratia rubidaea: a paradigm system for the three-isozyme gene family of enteric bacteria. Arch Biochem Biophys. 1992, 294: 147-153.PubMedView ArticleGoogle Scholar
- Maddock JR, Shapiro L: Polar location of the chemoreceptor complex in the Escherichia coli cell. Science. 1993, 259: 1717-1723.PubMedView ArticleGoogle Scholar
- Gu W, Zhao G, Eddy C, Jensen RA: Imidazole acetol phosphate aminotransferase in Zymomonas mobilis: molecular-genetic, biochemical and evolutionary analyses. J Bacteriol. 1995, 177: 1576-1584.PubMedPubMed CentralGoogle Scholar
- Whitaker RJ, Gaines CG, Jensen RA: A multi-specific quintet of aromatic aminotransferases that overlap different biochemical pathways in Pseudomonas aeruginosa. J Biol Chem. 1982, 257: 13550-13556.PubMedGoogle Scholar
- Silhavy TJ, Berman ML, Enquist LW, (Eds): Experiments with Gene Fusions. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory,. 1984Google Scholar
- Gibson F: Chorismic acid: purification and some chemical and physical studies. Biochem J. 1964, 90: 256-261.PubMedPubMed CentralView ArticleGoogle Scholar
- Cotton RGH, Gibson F: The biosynthesis of phenylalanine and tyrosine: enzymes converting chorismic acid into prephenic acid and their relationship to prephenate dehydratase and prephenate dehydrogenase. Biochim Biophys Acta. 1965, 100: 75-88.Google Scholar
- Chaudhuri S, Anton IA, Coggins JR: Shikimate dehydrogenase from Escherichia coli. Methods Enzymol. 1987, 142: 315-320.PubMedView ArticleGoogle Scholar
- Brickman E, Beckwith J: Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and transducing phages. J Mol Biol. 1975, 96: 307-316.PubMedView ArticleGoogle Scholar
- Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1006/abio.1976.9999.PubMedView ArticleGoogle Scholar
- Kessler K, Safrin M: The propeptide Pseudomonas aeruginosa elastase acts as an elastase inhibitor. J Biol Chem. 1994, 269: 22726-22731.PubMedGoogle Scholar
- Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press,. 1989Google Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685.PubMedView ArticleGoogle Scholar
- Ahmad S, Jensen RA: The evolutionary history of two bifunctional proteins that emerged in the purple bacteria. Trends Biochem Sci. 1986, 11: 108-112. 10.1016/0968-0004(86)90044-7.View ArticleGoogle Scholar
- Gu W, Williams D, Aldrich H, Xie G, Gabriel DW, Jensen RA: The AroQ and PheA domains of the bifunctional P-protein from Xanthomonas campestris in a context of genomic comparison. Microb Comp Genomics. 1997, 2: 141-158.PubMedView ArticleGoogle Scholar
- ExPASY Molecular Biology Server. [http://www.expasy.ch]
- Nishioka M, Demerec M, Eisenstark A: Genetic analysis of aromatic mutants of Salmonella typhimurium. Genetics. 1967, 56: 341-351.PubMedPubMed CentralGoogle Scholar
- Berry A, Bhatnagar RK, Jensen RA: Enzymic basis for leakiness of auxotrophs for phenylalanine in Pseudomonas aeruginosa. J Gen Microbiol. 1987, 133: 3257-3263.PubMedGoogle Scholar
- Walker GE, Dunbar B, Hunter IS, Nimmo HG, Coggins JR: Evidence for a novel class of microbial 3-deoxy-D -arabino-heptulosonate-7-phosphate synthase in Streptomyces coelicolor A3(2), Streptomyces rimosus and Neurospora crassa. Microbiology. 1996, 142: 1973-1982.PubMedView ArticleGoogle Scholar
- Corpet F: Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988, 16: 10881-10890.PubMedPubMed CentralView ArticleGoogle Scholar