Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, PO Box 14, 9750 AA Haren, The Netherlands
Correspondence
L. Dijkhuizen
L.Dijkhuizen{at}biol.rug.nl
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ABSTRACT |
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The GenBank accession numbers for the DAHP synthase encoding nucleotide sequences of A. methanolica presented in this paper are AY382157 (aroF) and AY382158 (aroG).
Present address: Organic-Chemistry Institute, University of Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.
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INTRODUCTION |
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The shikimate pathway and the Phe-specific pathway are controlled by three regulatory enzymes: 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase, chorismate mutase (CM) and prephenate dehydratase (PDT). DAHP synthase, the first enzyme of the shikimate pathway, is responsible for the condensation of the pentose phosphate pathway intermediate D-erythrose 4-phosphate and the glycolytic pathway intermediate phosphoenolpyruvate to DAHP. Carbon flow through the shikimate pathway, where tested in other microbes, is generally controlled by feedback inhibition of DAHP synthase activity and/or repression of DAHP synthase synthesis. DAHP synthase enzymes are present either as a monofunctional di- or tetrameric protein, e.g. in Escherichia coli (Ray & Bauerle, 1991; McCandliss et al., 1978
; Schoner & Herrmann, 1976
), or as a bifunctional protein exhibiting both DAHP synthase and CM activities, e.g. in Brevibacterium flavum (Sugimoto & Shiio, 1980
) and in A. methanolica (Euverink et al., 1995a
). Several organisms contain two or three isoenzymes of DAHP synthase, each displaying a specific feedback inhibition pattern.
Based on their highly divergent primary structures, two DAHP synthase families are distinguished. E. coli-type DAHP synthase enzymes have only been found in micro-organisms. Plant-type DAHP synthase proteins are mainly found in plants, but an increasing number of these enzymes are found in bacteria, e.g. in the aurachin-producing Gram-negative bacterium Stigmatella aurantiaca (Silakowski et al., 2000), and in the phenazine biosynthetic gene cluster of Pseudomonas aureofaciens (phzF). It has been speculated that the phzF gene product serves to bypass feedback-inhibited DAHP synthase protein(s) in P. aureofaciens to ensure sufficient intracellular levels of chorismate for phenazine production (Pierson et al., 1995
). Plant-type DAHP synthase enzymes are also found in actinomycetes; e.g. the Streptomyces avermitilis (BAC73797) (Ikeda et al., 2003
) and Streptomyces coelicolor (P80574, CAB38581) (Bentley et al., 2002
) genomes encode one and two (putative) plant-type DAHP synthase proteins, respectively. The Corynebacterium glutamicum genome encodes single copies of both DAHP synthase protein families (BAB99571, BAB98383), similar to the rifamycin producer A. mediterranei (AAK28148, AAC01718). The ORF encoding the plant-type DAHP synthase, located in the rifamycin biosynthetic gene cluster, is involved in aminoDAHP synthesis (August et al., 1998
).
Biochemical studies already have shown that two DAHP synthase isoenzymes and single CM and PDT enzymes are present in A. methanolica. DAHP synthase 1 (DS1) is a 160 kDa enzyme associated non-covalently with a dimeric CM protein, thus forming a heteromeric two-enzyme complex. The two enzyme activities can be separated by Q-Sepharose anion-exchange chromatography, yielding a dimeric CM protein with a fivefold reduced activity that is no longer feedback-inhibited by Phe and Tyr, and a 160 kDa DAHP synthase that is still feedback inhibition sensitive to its effectors Phe, Tyr and (most strongly) Trp (Euverink et al., 1995a).
Characterization of a leaky Phe auxotrophic mutant (GH141) of A. methanolica revealed that it had lost 90 % of the Phe aminotransferase activity, resulting in Phe-limited growth in mineral medium. Mutant GH141 expressed an additional Tyr-sensitive DAHP synthase activity (DS2), accompanied by a strongly elevated CM activity. In mutant GH141 the CM protein was not associated with DS1 activity, but occurred as a separate dimeric protein (Euverink et al., 1995a). Supplementing Phe to the growth medium of GH141 restored wild-type activity levels of both CM and DAHP synthase. An o-fluoro-DL-phenylalanine-resistant mutant (oFPhe83) was subsequently characterized that showed high levels of DS2 and dimeric CM activity, in both the presence and absence of Phe, suggesting a regulatory mutation that derepressed the synthesis of both proteins (Euverink et al., 1995a
). Thus, whereas DS1 activity is sensitive to Phe, Tyr and (most strongly) Trp feedback inhibition, DS2 activity is sensitive to Tyr (but not Phe) feedback inhibition whereas its synthesis is sensitive to feedback repression mediated by Phe.
Prephenate dehydratase (PDT) enzymes occur as monofunctional proteins in Gram-positive bacteria or as bifunctional proteins (P-proteins) in Gram-negative bacteria (Bentley, 1990). PDT enzymes are generally sensitive to feedback regulation by Phe and/or Tyr (Bentley, 1990
). PDT of A. methanolica is allosterically inhibited by Phe and activated by Tyr (de Boer et al., 1989
; Euverink et al., 1995b
). Previously, we reported characterization of the A. methanolica pdt gene (Vrijbloed et al., 1995
).
Here we report the isolation and characterization of a number of spontaneous A. methanolica PDT mutants insensitive to Phe feedback inhibition and Tyr feedback activation, providing new information about amino acid residues involved in PDT allosteric control. Furthermore, we report the molecular and biochemical characterization of a second DAHP synthase in A. methanolica and molecular characterization of both DAHP synthases of A. methanolica, revealing that this organism contains a single representative of both currently recognized families of DAHP synthase enzymes. We believe this to be the first report of a plant-type DAHP synthase that forms a protein complex with a CM, thereby stimulating CM activity and making it sensitive to feedback inhibition.
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METHODS |
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Preparation of extracts and enzyme assays.
Cells were washed in buffer (50 mM Tris/HCl pH 7·5) and disrupted by three passages through a French pressure cell at 140 MPa. Unbroken cells and debris were removed by centrifugation of the lysate at 40 000 g for 30 min at 4 °C and the supernatant was used for enzyme assays. Unless otherwise stated enzyme assays were performed at 37 °C.
PDT (EC 4.2.1.51) activity was assayed by measuring phenylpyruvate formation. The reaction mixture (0·5 ml) contained 1 mM potassium prephenate, 50 mM Tris/HCl pH 7·5 and protein. At appropriate time intervals 0·5 ml 2·0 M NaOH was added and the absorbance of phenylpyruvate was measured at 320 nm (320[phenylpyruvate]=17·5x103 M-1 cm-1) (Patel et al., 1977
). CM (EC 5.4.99.5) activity was assayed by measuring the amount of prephenate formed after its conversion to phenylpyruvate (Dopheide et al., 1972
). The reaction mixture (0·1 ml) contained 50 mM Tris/HCl, pH 7·5, 2·0 mM chorismate and protein. After 10 min, 10 µl 4·5 M HCl was added and the reaction mixture was incubated for 15 min at 37 °C. After addition of NaOH (890 µl of a 1·58 M solution) the phenylpyruvate formed was determined by measuring A320. DAHP synthase (EC 2.5.1.54) activity was assayed as described by de Boer et al. (1989)
. Phe, Tyr and Trp inhibition/activation of PDT, CM and DAHP synthase activity was determined in the presence of 1 mM of each amino acid (Euverink et al., 1995a
, b
).
DNA sequence analysis.
Nucleotide sequencing was done using dye-primers in the cycle sequencing method (Murray, 1989) with the thermosequenase kit RPN 2538 from Amersham Pharmacia Biotech. The samples were run on the ALF-Express sequencing robot. Analysis of nucleotide sequence was done using CloneManager version 4.01. Protein sequence comparisons were performed using the facilities of the BLAST server (Altschul et al., 1990
) at NCBI (National Library of Medicine, Washington, DC, USA).
PCR primers used to clone aroG.
Using alignments of plant-type (putative) DAHP synthase proteins (see Fig. 3), degenerate primers were designed on the basis of two conserved motifs: PLDS1 [5'-GG(G/C)(A/C)G(G/C)ATCGC(G/C)GG(G/C)CA(G/A)-3'] and PLDS2 [5'-(G/C)GTGTT(G/C)CCGTGCAT(G/C)GG-3']. PCR experiments with these primers, using A. methanolica DNA as template, yielded a single DNA fragment of approximately 730 bp with strong sequence similarity to plant-type DAHP synthase genes. Specific primers PLDS3 and PLDS4 were designed (Fig. 3
), and used to screen an A. methanolica genomic library.
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Gene library screening for the aroG gene was performed by PCR, using primers PLDS3 (5'-TCGCACTTCCTGTGGAT-3') and PLDS4 (5'-TGCCAGATGACCTTGTG-3'). Plates with 100 gene library transformants were replica-plated. Subsequently, all cell material from the original plate was resuspended in 1 ml LB medium, from which plasmid DNA was isolated and subjected to PCR analysis. Colonies originating from plates with a positive outcome were subjected to individual PCR analysis. This resulted in identification of a positive clone containing an insert of approximately 8 kb.
Southern hybridization of aroF.
Digested chromosomal DNA from A. methanolica was separated on a 0·8 % (w/v) agarose gel and blotted onto a high-bond nylon membrane (Qiagen), via an alkaline transfer method (Sambrook et al., 1989). Southern hybridization was performed at 65 °C, using the entire aroF gene (for DS2) of A. methanolica as a probe. Radioactive probe labelling was performed with the high prime DNA labelling kit from Boehringer Mannheim. Following hybridization the membrane was washed at 65 °C with 2x SSC (1x SSC is 0·15 M NaCl and 0·015 M sodium citrate) containing 0·5 % (w/v) SDS for 10 min, twice with 1x SSC containing 0·5 % (w/v) SDS at room temperature and three times with 0·3x SSC containing 0·5 % (w/v) SDS at room temperature.
Estimation of the molecular mass of native proteins.
The molecular masses of AroF and AroG were estimated by loading E. coli cell-free extracts on a Superdex-200 column (XK 16/60) equilibrated with Tris/HCl buffer (pH 7·5). Bio-Rad gel filtration standard proteins (670, 158, 44 and 17 kDa proteins) were used as standards.
Analytical methods.
Protein concentrations were determined with the protein determination kit from Bio-Rad, using bovine serum albumin as standard (Bradford, 1976).
Accession numbers.
The DAHP synthase encoding nucleotide sequences of A. methanolica presented in this paper were entered into GenBank under accession numbers AY382157 (aroF) and AY382158 (aroG). The accession number of the A. methanolica pdt gene sequence is Q44104.
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RESULTS |
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A. methanolica wild-type PDT activity is inhibited up to 70 % by 1 mM Phe, and stimulated by a factor of two by a similar amount of Tyr (Table 2) (Euverink et al., 1995b
). Of 76 colonies tested, seven had (completely) lost Phe feedback inhibition sensitivity, and five of those seven mutants had also completely lost the stimulatory effect of Tyr. Similar results were obtained for PDT enzymes in two previously (Euverink et al., 1995b
) isolated FPhe-resistant A. methanolica mutants, strains pFPhe32 and oFPhe84. None of these nine strains carrying PDT mutants were affected in CM sensitivity to Phe inhibition. No PDT mutants were detected that had lost the stimulatory effect of Tyr only.
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CM only displays its feedback inhibition sensitivity when bound within the CMDAHP synthase enzyme complex (with DS1; see below) (Euverink et al., 1995a). The inhibitor-binding domain involved in this phenomenon thus may be located on the DAHP synthase moiety. In this situation, two types of CM and DAHP synthase feedback inhibition resistant mutants may occur: (I) a DS1 mutation resulting in loss of feedback inhibition sensitivity of both CM activity and DS1 activity, and (II) a regulatory mutation leading to derepression of (Phe feedback inhibition insensitive) DS2 and CM synthesis, similar to mutant oFPhe83 (Euverink et al., 1995a
). The two isolated mutant strains with deregulated CM enzymes were therefore tested for their DAHP synthase activities and feedback inhibition patterns. Mutants pFPhe4 and pFPhe25 both possessed 910-fold higher DAHP synthase activity levels. Similar to mutant oFPhe83, this DAHP synthase activity was most strongly feedback-inhibited by Tyr, indicating the presence of (derepressed) DS2 activity (Table 3
). Screening of the remaining 74 pFPhe-resistant mutant strains did not yield a single mutant with deregulated DS1 activity.
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PDT sequence alignments
Sequence alignments revealed that A. methanolica PDT shares considerable similarity with other (monofunctional) actinomycete PDT sequences (Fig. 2). Also the PDT-encoding domains of P-proteins aligned well with A. methanolica PDT, albeit with lower relative sequence similarity.
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Expression of aroF was stimulated by one order of magnitude when 0·5 M sorbitol was added to the LB growth medium as a compatible solute (Table 3). The amount of AroF present in extracts of cells grown on LB with 0·5 M sorbitol was estimated to be about 20 % of total protein as observed on SDS-PAGE gels (data not shown).
During PCR amplification of the aroF gene, a PCR fragment was isolated with an opal stop codon mutation at nucleotide position 907 of the ORF. Expression of this mutated aroF (pHK276-0), encoding a protein with a subunit molecular mass of 31·5 kDa instead of 37·8 kDa, yielded a protein insensitive to feedback inhibition. Rather, Phe and Trp now had a stimulatory effect on the activity of the truncated protein (Table 3).
Cloning and characterization of a second DAHP synthase gene
Attempts to clone the DS1 gene from chromosomal DNA of A. methanolica in Southern hybridization experiments using aroF as a probe failed (data not shown). Using PCR screening, we therefore searched for a plant-type DAHP synthase gene in A. methanolica.
DNA sequence determination of 4·5 kb of the insert of the positive clone selected (see Methods), revealed an ORF of 1391 bp potentially encoding a 50 687 kDa protein, showing a high degree of sequence similarity toward other plant-type DAHP synthase proteins (Fig. 3). Heterologous expression of this ORF, designated aroG, was achieved in E. coli by cloning the gene into the expression vector pET3b (pHK287). DAHP synthase activity in cell extracts of E. coli strain BL21(DE3)/pHK287 was very sensitive to Trp feedback inhibition, as observed for DS1 of A. methanolica (Table 3
, wild-type situation). The amount of AroG present in extracts of cells of the heterologous host was estimated to be approximately 15 %, as observed on SDS-PAGE gels (data not shown).
Gel filtration analysis of A. methanolica AroF and AroG
Extracts of mutant oFPhe83 expressed DS2 DAHP synthase protein in a monomeric form (Euverink et al., 1995a). The native molecular masses of both A. methanolica DAHP synthase proteins expressed in E. coli were determined by gel filtration chromatography on a Superdex-200 column. Elution times of the AroF and AroG activity peaks corresponded to molecular masses of 160 and 200 kDa, respectively (data not shown). In view of their subunit molecular masses (37·8 and 50·7 kDa, respectively), these results indicate that both proteins are expressed as tetramers in E. coli. No (significant) monomeric DAHP synthase activity could be determined in either of the extracts, corresponding to the DS2 activity found earlier in A. methanolica mutants GH141 and oFPhe83 (Euverink et al., 1995a
).
Effects of AroG on CM activity and feedback inhibition sensitivity
Mutant strain GH141 displayed a strongly increased level of deregulated CM activity, not associated with a DAHP synthase enzyme. The dimeric CM activity of this mutant could be activated and its feedback control restored by addition of gel filtration fractions containing DAHP synthase activity (Euverink et al., 1995a).
Titration of Superdex-200 gel filtration fractions containing DS1 AroG (purity approx. 60 %) to cell extracts of mutant GH141 stimulated CM activity by a factor of 5, also restoring its feedback inhibition sensitivity (Fig. 4). These effects were not observed in titration experiments with DS2 AroF to cell extracts of GH141.
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DISCUSSION |
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All other mutations found to affect A. methanolica PDT feedback inhibition control are located in the vicinity of but not within the highly conserved GALV and ESRP regions (E. coli P-protein residues 309312 and 329332, respectively) (Fig. 2). Mutations in the hydrophobic GALV region, decreasing its hydrophobic nature or introducing large side chains, impaired Phe binding in the E. coli P-protein (Pohnert et al., 1999
). Conserved residues within the GALV and ESRP regions may also be important in Phe and Tyr binding in A. methanolica PDT, but mutations in these residues may well be detrimental for PDT activity due to incorrect protein folding. Obviously, such mutations will not be found when screening for fully active, deregulated PDT mutants.
The A. methanolica PDT mutant H227R is located near the ESRP region; mutants A198P, A198V, N200K and L209F are all located near the GALV region. A198, which is not a conserved residue, may be part of the hydrophobic binding pocket and the introduction of the more bulky Pro and Val side chains may prevent Phe and Tyr binding. The Lactococcus lactis and Aquifex aeolicus putative PDT proteins, however, do contain a Pro and a Val residue at their equivalent A198 positions, respectively. These proteins have not been characterized yet, however, and their sensitivity to feedback inhibition by Phe remains to be studied. It is unlikely that the charged N200, which is conserved in various PDT proteins, is (directly) involved in Phe (or Tyr) side chain binding. Also binding of the carboxyl group of the effector molecule is unlikely, since it is still activated by Tyr. The P-protein of E. coli contains a Gln at this position, which was shown to be essential for feedback regulation (Nelms et al., 1992). The well-conserved L209 may be part of a hydrophobic pocket, involved in binding of the Phe (and Tyr) side chain. Why Phe feedback inhibition resistant mutant L209F is still capable of Tyr binding remains unclear.
Only two CM and DAHP synthase mutants were identified among the pFPhe-resistant strains, both with derepressed CM and DS2 activity levels. We failed to clone the A. methanolica gene encoding CM. Sequence analysis of the surrounding regions of the aroF and aroG genes revealed that the CM gene is not organized in an operon with one of these DAHP synthase genes. The derepression of both activities suggests that a common (unidentified) transcriptional regulator of both genes has been mutated. No AroG feedback-inhibition-resistant mutants were isolated, which also would have resulted in a deregulated CM activity. Mutant selection was directed toward active proteins, and deregulated AroG mutants may possess no or only low activities. Such mutants therefore may have escaped detection in the selection procedure used.
Several plant-type DAHP synthases have been identified in actinomycetes, e.g. in Streptomyces species (Walker et al., 1996). This type of DAHP synthase is often involved in the biosynthesis of secondary metabolites, such as the ansamycin antibiotic rifamycin in A. mediterranei (Yu et al., 2001
), phenazine in P. aureofaciens (Pierson et al., 1995
), aurachin in Stigmatella aurantiaca (Silakowski et al., 2000
), chloramphenicol in Streptomyces venezuelae (He et al., 2001
) and ansatrienin and naphthomycin in Streptomyces collinus (Chen et al., 1999
). In contrast, in Xanthomonas campestris a plant-type enzyme functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis (Gosset et al., 2001
).
Expression of both A. methanolica DAHP synthase proteins in E. coli was successful. Addition of 0·5 M sorbitol even further increased DAHP synthase activity by a factor of 8. The inhibition pattern of AroG was similar to that of DS1 activity, with Trp as its main effector, and that of AroF was similar to DS2 activity, with Tyr showing the strongest inhibition.
Gel filtration experiments show that AroF is expressed in E. coli as a tetramer. The protein responsible for DS2 activity found in mutant A. methanolica strain oFPhe83, however, was monomeric with a molecular mass of approximately 42 kDa. Possibly, AroF expressed in E. coli is assembled as a tetramer, while it remains a monomer in its original host. PCR fragment analysis did not indicate the presence of a second plant-type DAHP synthase gene, since several cloned PCR fragments were identical in DNA sequence. Furthermore, feedback inhibition patterns of DAHP synthase activities in wild-type and E. coli/pHK276 were similar (Table 3).
From the CMDAHP synthase titration experiments we conclude that AroG is associated with CM to form a heterohexameric enzyme complex. AroG clearly activates CM activity up to a factor of 5 and restores its feedback sensitivity to Phe and Tyr, as observed in the A. methanolica wild-type strain. The close association of the AroG and CM proteins, and the feedback inhibition pattern of AroG, show that AroG activity supports aromatic amino acid biosynthesis in A. methanolica. However, AroF activity also potentially makes an important contribution, becoming derepressed under Phe limiting conditions (this study; Euverink et al., 1995a).
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Received 16 May 2003;
revised 25 July 2003;
accepted 13 August 2003.
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