The Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield S10 2TN, UK1
Author for correspondence: John R. Guest. Tel: +44 114 2224406. Fax: +44 114 2728697. e-mail: j.r.guest{at}sheffield.ac.uk
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ABSTRACT |
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Keywords: aconitase, ironsulphur proteins, propionate metabolism, YbhJ
Abbreviations: Acn, aconitase; c-Acn, cytoplasmic aconitase; mit-Acn, mitochondrial aconitase; AcnC, the residual Acn activity of an AcnAB-null strain; IRP, iron regulatory protein; PrpD, 2-methylcitrate dehydratase; UTR, untranslated region
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INTRODUCTION |
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Biochemical and enzymological studies including studies with acn mutants and acnlacZ reporter fusions have established that AcnB is the major citric acid cycle enzyme, synthesized during exponential growth and adapted for this role by performing better at high substrate concentrations (Gruer et al., 1997b ; Cunningham et al., 1997
; Jordan et al., 1999
). In comparison, AcnA is a more robust stationary-phase enzyme that is specifically induced by iron and oxidative stress, presumably to maintain citric-acid-cycle functions under oxidative stress conditions. The aerobic growth of acnA mutants is essentially unimpaired in minimal medium with glucose and other substrates including pyruvate and acetate, indicating that the lesion is fully complemented by the acnB gene (Gruer et al., 1997b
). In contrast, acnB mutants are severely impaired, and although the growth defect is partly reversed by adding glutamate, acetate cannot be used even with a glutamate supplement. However, acnAB double mutants are even more debilitated, indicating that the acnB lesion is complemented to a minor extent by the acnA gene.
The bifunctional c-Acn/IRP1 proteins of higher organisms lose their ironsulphur clusters and catalytic activity in response to iron starvation or oxidative stress and the apo-proteins then function as site-specific mRNA-binding proteins (Beinert et al., 1996 ; Hentze & Kuhn, 1996
). They bind to specific stemloop structures (iron-responsive elements, IRE) in the 5' or 3' UTR of relevant transcripts and either block translation or increase transcript stability (and hence translation), respectively. Recently, the apo-proteins of both E. coli aconitases (apo-AcnA and apo-AcnB) have been shown to interact with sequences in the 3' UTR of acnA and acnB mRNA, thereby increasing mRNA stability and hence Acn synthesis, in response to oxidative disassembly of the ironsulphur clusters (Tang & Guest, 1999
). The Bacillus subtilis aconitase (CitB) has likewise been shown to bind to rabbit ferritin IRE and to analogous sequences in relevant B. subtilis operons (Alen & Sonenshein, 1999
). There is also evidence that aconitase performs a regulatory role in virulence factor production in Pseudomonas aeruginosa and Xanthomonas campestris (Somerville et al., 1999
; Wilson et al., 1998
).
The purpose of the present work was to identify the source(s) of the aconitase activity retained by a derivative of E. coli W3110 (JRG3259) in which the acnA and acnB genes had each been totally inactivated by internal deletion and antibiotic-cassette insertion (Gruer et al., 1997b ). This residual aconitase activity, designated AcnC, amounted to approximately 5% of total wild-type activity. Two potential candidates, PrpD and YbhJ, were identified by partial purification of the residual activity and database searching, respectively. Evidence is presented to show that the AcnC activity can be attributed to the broad substrate specificity of PrpD, a 2-methylcitrate dehydratase that functions in the 2-methylcitrate pathway of propionate utilization.
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METHODS |
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Aconitase assay, reactivation and protein analysis.
Aconitase activity was assayed spectrophotometrically at pH 7·4 and 37 °C by following the conversion of DL-isocitrate (20 mM) to cis-aconitate at 240 nm using an absorption coefficient of 3·6 mM-1 cm-1 (Kennedy et al., 1983 ). One unit (U) corresponds to 1 µmol cis-aconitate formed min1; activities were routinely measured before and after reactivation by adding ammonium ferrous sulphate (0·5 mM), DTT (5 mM) and Tris/HCl (50 mM; pH 8·0) to the enzyme solution for 30 min at 0 °C. In specificity studies isocitrate was replaced by other potential substrates using concentrations of 20 mM for hydroxyacids and 0·2 mM for potentially hydratable double-bonded substrates under the same conditions as the standard assay. When required, 2-methylcitrate was generated enzymically by incubating propionyl-CoA (0·6 mM), oxaloacetic acid (0·6 mM) and Tris/HCl (20 mM pH 8·0) with porcine citrate synthase (1 U) in a final volume of 1·0 ml for 1 h at 37 °C. Reactions were stopped by heating for 10 min at 70 °C and clarified in a microfuge (13000 r.p.m., 10 min). Parallel reactions with acetyl-CoA instead of propionyl-CoA were used to generate citrate, and reactions lacking each substrate served as controls. Although porcine citrate synthase is a poor 2-methylcitrate synthase compared to the Thermoplasma acidophilum enzyme (Gerike et al., 1998
), the former was found to use propionyl-CoA at 2% of the rate observed with acetyl-CoA and both enzymes gave comparable products under the conditions used. The porcine enzyme was used routinely because it is more readily inactivated. 2-Methylcitrate was converted (presumably to 2-methyl-cis-aconitate) by adding purified PrpD (0·06 U) directly to the clarified reaction products (and controls), incubating at 37 °C until the absorbance at 240 nm stopped increasing (10 min approx.), and then heating and clarifying as before.
Ironsulphur clusters were also reconstituted by a NifS-mediated procedure (Green et al., 1996 ). Purified PrpD (520 nmol in 0·1 M Tris/HCl, pH 7·4) was equilibrated in an anaerobic workstation (Don Whitely Scientific, Mk3) at 25 °C for 16 h before adding DTT (5 mM), L-cysteine (1 mM), (NH4)2Fe(SO4)2 (10 mol per mol of PrpD) and NifS (1 µM), in 1 ml final volume. The reaction mixtures were sealed in quartz cuvettes and changes in the absorbance in the range 240900 nm were monitored using a Unicam UV4 spectrophotometer; mixtures lacking NifS or PrpD were used as controls. For subsequent analysis, excess iron and sulphur were removed under anaerobic conditions by loading on an Ether 650S column (9x12 mM) washing with high-salt buffer (2 ml 10 mM Tris/HCl, pH 8·0, containing 1 M NaCl) and eluting with low-salt buffer (2 ml 10 mM Tris/HCl, pH 8·0, containing 10 mM NaCl).
Partial purification of AcnC from an unamplified source.
AcnC activity was partially purified from the AcnAB-null strain (JRG3259; acnA::kanR acnB::tetR) grown with vigorous aeration at 37 °C for 812 h in six 2 l flasks each containing 500 ml L broth supplemented with appropriate antibiotics and inoculated to give a starting OD600 of 0·1. The bacteria were harvested, resuspended at 4 °C in 0·1 M Tris/HCl, pH 7·4 (buffer A), containing 10 mM benzamidine, disrupted by three passes through a French pressure cell (15000 p.s.i., 103·5 kPa), and debris removed by centrifugation (25000 g for 30 min). The cell-free extracts were fractionated by dye-affinity chromatography, based on the methods used for AcnA and AcnB (Bennett et al., 1995 ; Bradbury et al., 1996
), using a Procion Red column (20x90 mM) previously equilibrated with buffer A. Bound protein was eluted at 0·5 ml min1 with a 50 ml linear gradient of 01 M citrate in buffer A. Fractions were tested before and after reactivation and active fractions (eluted at 100200 mM citrate) were pooled, desalted by dialysis in 2 l buffer A (overnight), and then applied to a Procion Yellow column (151x40 mM; also equilibrated with buffer A). Active fractions were again eluted at approximately 100200 mM citrate using the same gradient (01 M citrate), pooled and stored at -20 °C after desalting.
Purification of PrpD and YbhJ from genetically amplified strains.
The PrpD and YbhJ proteins were purified from strains containing the corresponding ptac-85 expression plasmids (pGS1575 and pGS1576) in the AcnAB-null host (JRG3259); see Table 1. Cultures were grown with vigorous aeration in six 2 l baffled flasks each containing 500 ml L broth. After 1 h at 37 °C they were transferred to 25 °C, IPTG (30 mg l-1) was added and growth was continued for a further 812 h. The overproduced PrpD was isolated as described above for AcnC; the aconitase activity and enrichment in the SDS-PAGE profile were used to monitor the purification. The YbhJ protein was partially purified to 50% purity by dye-affinity chromatography as for PrpD except that NaCl (1 M) was used in the salt gradients. The protein eluted at 100200 mM NaCl and purification was monitored solely by SDS-PAGE because no aconitase activity was associated with the enriched fractions.
PAGE, Mr determination and other analytical procedures.
PAGE was according to Laemmli (1970) using denaturing conditions (0·1% SDS and 10 or 15% acrylamide), or non-denaturing conditions (no SDS and 515% acrylamide). The Mr of PrpD was deduced from denaturing SDS-PAGE, native gel mobilities, and gel filtration using a Sephacryl S200 HR column (20x330 mM) equilibrated and eluted with buffer A containing 1 M citrate. The column was calibrated with standard proteins: ß-amylase (Mr 200000), alcohol dehydrogenase (150000), bovine serum albumin (66000), carbonic anhydrase (29 000) and cytochrome c (12400). The N-terminal amino acid sequences were determined by Edman degradation using an Applied Biosystems Protein Sequencer with samples from Coomassie-blue-stained electro-blots of SDS-PAGE gels. For Western blotting, samples containing 15 µg protein fractionated by SDS-PAGE were transferred to Hybond C (Amersham) in a Bio-Rad Transblot Electrophoretic Transfer Cell and the blots were immuno-stained using polyclonal antisera according to Gruer et al. (1997b
). Protein concentrations were determined by the Bio-Rad micro-assay procedure. Iron contents were assayed as described by Woodland & Dalton (1984)
and sulphur contents were assayed according to Beinert (1983)
.
DNA manipulation and methods.
DNA was prepared and manipulated by standard procedures (Sambrook et al., 1989 ). Expression plasmids containing ybhJ and prpD were constructed by PCR-amplification of these genes from W3110 chromosomal DNA with the simultaneous incorporation of flanking NcoI and SalI restriction sites (underlined). The oligonucleotides used in the PCR amplification and cloning of prpD and ybhJ sequences were: prpD forward primer, GCGCACTTCACCATGGCAGATATCTGG; prpD reverse primer, GCCTTATCTGGTCGACAGATTCGATGCG; ybhJ forward primer, CTTTCTGCGCACTTCACCATGG-CAGATATCTGGAGC; ybhJ reverse primer, GCCTTATCTGGTCGACAGATTCGATGCGATTCG. The PCR reactions each contained 200 ng template DNA, 100 pmol each primer and 1 U Taq DNA polymerase (Promega). The major amplified product in each reaction was purified and cloned in ptac-85 (Marsh, 1986
) as NcoISalI fragments. Plasmids from ApR transformants of DH5
were screened for inserts of the desired size and the constructions were confirmed by repeat PCR with the same primers followed by restriction digestion and DNA sequencing of the amplified products. Representative plasmids were designated pGS1575 (prpD+) and pGS1576 (ybhJ+) and transferred to JRG3259. Synthesis of RNA and gel retardation analysis to determine mRNA binding properties was according to Tang & Guest (1999)
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Mutant construction.
An acnA acnB ybhJ triple mutant was constructed by sequential P1vir-mediated transduction of acnA::kanR (from JRG2789) and acnB::tetR (from JRG3258) into KS302, a strain that lacks the galybhJbio region. The authenticity of the final mutant JRG4195 (acnA acnB galbio) was confirmed by PCR with primers flanking the deletion. To provide an isogenic ybhJ+ control strain, the galybhJbio region was transferred from W3110 to JRG4195 by P1vir transduction and selection for Gal+ Bio+ colonies. Restoration of the wild-type galybhJbio region was confirmed by nutritional tests for Gal+ and Bio+ and by DNA sequencing with a representative strain designated JRG4629. A prpD mutation was generated by direct linear DNA transformation of strain DY329 according to Yu et al. (2000)
. Two PCR primers containing 40 bp prpD sequence and 20 bp camR cassette (italic) were synthesized (forward primer ATGTCAGCTCAAATCAACAACATCCGCCCGGAATTTGATC-ATGGAGAAAAAAATCACTGG; reverse primer TTAAATGACGTACAGGTCGAGATACTCATTGACCGGCATC-TTACGCCCCGCCCTGCCACT) and used with pACYC184 as template to amplify DNA molecules containing the entire camR cassette flanked by prpD sequences. This amplified DNA promoted the replacement of 885 bp (90%) of the chromosomal prpD gene by the camR cassette in CmR transformants of DY329. The location of the prpD::camR lesion in a representative transformant (JRG4627) was confirmed by the size and restriction profile of the corresponding PCR-amplified segment of chromosomal DNA. The prpD::camR lesion was then transferred to JRG3259 (acnA acnB) by P1vir-mediated transduction to generate the corresponding triple mutant, JRG4628 (acnA acnB prpD::camR), where the accuracy of the construction was reconfirmed by analysing the PCR-amplified DNA and sequencing across both prpDcamR junctions.
Materials.
Samples of purified enzymes were kindly provided by Dr Y. Tang (E. coli aconitases, AcnA and AcnB) and Dr E. Ralph (Azotobacter vinelandii cysteine desulphurase, NifS). Porcine heart and Thermoplasma acidophilum citrate synthases were purchased from Sigma-Aldrich UK, as were acetyl-CoA, propionyl-CoA and substrates used in specificity tests.
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RESULTS |
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Sequence relationships between aconitase and two potential AcnC candidates
Searching the complete E. coli genome (Blattner et al., 1997 ) revealed one potential AcnC candidate, YbhJ, encoded by a gene located in the ybhDHIJybhCatt
ybhBbioA region at 17·4 min (800 kb) in the E. coli chromosome. The predicted YbhJ sequence (761 amino acids, 81505 Da) clearly indicated that YbhJ is a member of the aconitase protein family having the same domain organization as porcine mit-Acn and E. coli AcnA and sequence identities of 24% and 22%, respectively (Fig. 2
). Conservation of the three cysteine residues that serve as ligands for ironsulphur clusters in Acn proteins indicated that YbhJ might likewise assemble such clusters. However, only 12 of the other 17 Acn active-site residues are conserved, suggesting that YbhJ may not exhibit aconitase activity although it could have a different catalytic specificity. The size predicted for YbhJ (81505 Da) is significantly smaller than typical aconitases and it is also considerably larger than the AcnAB cross-reacting component (Mr 58000 approx.) associated with the AcnC activity that was partially purified from JRG3259 (see above).
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Overexpression and inactivation of AcnC candidates (PrpD and YbhJ)
To establish whether either of the AcnC candidates (PrpD and YbhJ) contribute to the residual aconitase activity in JRG3259 (acnA acnB) the effects both of overexpressing and inactivating the prpD and ybhJ genes on the AcnC activities of AcnAB-null strains were investigated. The relevant coding regions were PCR-amplified and cloned in ptac-85 to generate two expression plasmids, pGS1575 (ptac-85 prpD+) and pGS1576 (ptac-85 ybhJ+); see Methods. The protein profiles confirmed that the synthesis of both proteins in transformants of JRG3259 is greatly enhanced by IPTG (Fig. 3). Enzymological tests with IPTG-induced transformants of DH5
and JRG3259 further showed that prpD+ overexpression increased the combined aconitase and AcnC activities of the Acn+ host by 3542% and the AcnC activity of the AcnAB-null host by
26-fold, whereas ybhJ+ overexpression had no significant effect in either host (Table 3
). These results supported the view that PrpD might be responsible for the AcnC activity in JRG3259, but PrpD overproduction would not complement the growth phenotype of the AcnAB-null strain.
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Inactivation of PrpD was achieved by constructing a prpD::camR derivative of JRG3259 (acnA acnB) designated JRG4628 (acnA acnB prpD::camR) in which most of the chromosomal prpD gene is replaced by a camR cassette; see Methods. The aconitase specific activities in cell-free extracts of the double mutant, 0·020 U (mg protein)-1, were lowered to 0·002 U (mg protein)-1 in the triple mutant, indicating that disruption of the prpD gene virtually eliminates the residual aconitase activity (AcnC) of the AcnAB-null strain. These observations strongly indicate that PrpD is the major source (and probably the sole source) of AcnC activity. It is also relevant to note that growing the AcnAB-null strain in the presence of added propionate (1050 mM) increased the AcnC activity by up to 1·8-fold [0·036 U (mg protein)-1 with 40 mM propionate]. However, propionate was not normally added to growth media, so that the AcnC activity would not be masked or otherwise affected by specific propionate-inducible enzymes.
Purification, properties and metabolic function of PrpD
Although attempts to purify AcnC from an unamplified source (JRG3259; acnA acnB) failed to identify the enzyme unequivocally, they did provide valuable clues leading to the conclusion that PrpD is the source of AcnC activity. Further studies on PrpD were made with protein purified from a strain containing the prpD expression plasmid (pGS1575) in an AcnAB-null host (JRG3259). A typical purification producing PrpD protein that was judged to be 97% pure by quantitative densitometry (Fig. 1b), with a yield of 32 mg per litre of culture, is outlined in Table 2
. No decline in specific activity was observed, presumably because inactivated enzyme represents a much smaller proportion of the total protein recovered from the highly enriched starting material. The N-terminal amino acid sequence of the purified protein, XAQINNIRPEFD, was identical to that predicted for PrpD assuming that the unidentified residue (X) is serine and the initiating formylmethionine residue is removed post-translationally. The purified PrpD protein cross-reacted strongly with anti-AcnA and anti-AcnB sera (Fig. 1b
), providing further evidence to suggest that the cross-reacting component in the AcnC partially purified from the unamplified source is PrpD (Fig. 1a
). Gel filtration and native gel electrophoresis indicated that purified PrpD is a monomeric protein of Mr approximately 54000, which is consistent with the predicted Mr of 53821 for the 482-residue protein lacking the N-terminal methionine residue. The AcnC-like activity of PrpD exhibited the same pH profile (optimum range 7·08·0), stability (half-life of 30 min at 50 °C) and failure to use cis-aconitate as a substrate (see below), as the AcnC of cell-free extracts of the AcnAB-null strain (data not shown). As isolated, purified PrpD contained up to 1·26 atoms of iron and 1·4 atoms of acid-labile sulphur per monomer but very little activity, and after treatment with Fe2+ and DTT these values rose to 2·5 and 1·8 (respectively) and activity was restored, suggesting that PrpD contains an ironsulphur cluster that is essential for maximum activity. Reconstitution of the ironsulphur cluster was monitored spectrophotometrically under anaerobic conditions using cysteine desulphurase (NifS) as the sulphide generator (see Methods). As shown in Fig. 4(a)
, the spectrum acquired a broad absorbance band around 420 nm. After removing excess iron and sulphur under anaerobic conditions, the respective iron and acid-labile sulphur contents were 2·3 and 1·8 atoms per monomer. On exposure to air the 420 nm absorbance declined steadily over an 8 h period (Fig. 4b
) indicating that the ironsulphur clusters are unstable under aerobic conditions. It was therefore concluded that PrpD can acquire one [2Fe2S] cluster per monomer, which is essential for maximum catalytic activity.
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The prpD gene, like other genes in the prpRBCDE operon, has been shown to be required for propionate catabolism via the methylcitrate cycle in S. typhimurium (Horswill & Escalante-Semerena, 1999a , b
). The E. coli genome possesses an analogous prp operon but E. coli cannot use propionate as a sole carbon source without prior adaptation, possibly involving one or more unidentified mutations. Nevertheless, the prpB and prpC genes have been shown to encode two functional enzymes, 2-methylisocitrate lyase and 2-methylcitrate synthase, respectively (Textor et al., 1997
; Gerike et al., 1998
; Brock et al., 2001
). It has been further suggested that prpD might encode a methylaconitase, primarily because of its location in the prp operon and the known functions of the neighbouring genes (Horswill & Escalante-Semerena, 1999a
, b
; Brock et al., 2001
). Here, the substrate specificity of purified and reactivated E. coli PrpD was tested using a range of potential substrates including 2-methylcitrate (Table 4
). The latter was synthesized by preincubating propionyl-CoA and oxaloacetate with citrate synthase but not purified or subjected to isomer analysis (see Methods). The results showed that PrpD is capable of introducing a double bond into all nine of the hydroxyacids tested (Table 4
). Moreover, 2-methylcitrate proved to be a better substrate than citrate and citrate generated enzymically in directly comparable control reactions. This is consistent with the possibility that 2-methylcitrate is the primary substrate for PrpD. The preference for 2-methylcitrate relative to citrate was also exhibited by cell-free extracts of the AcnAB-null mutant, JRG3259 (Table 4
). In addition it was observed that activity with both citrate and 2-methylcitrate was abolished by the prpD lesion of JRG4628 (acnA acnB prpD). These observations further confirm that the AcnC activity of the acnAB double mutant is due to the broad substrate specificity of PrpD and its consequent ability to dehydrate citrate and isocitrate as well as 2-methylcitrate.
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Related observations with AcnA, AcnB and YbhJ
In reciprocal substrate specificity studies, AcnA and AcnB were tested for their ability to use enzymically generated citrate, 2-methylcitrate and 2-methyl-cis-aconitate, assuming that the latter is the product of sequential citrate synthase and PrpD preincubations and that the reactions are not impaired by other materials in the unpurified substrate (Table 5). The two aconitases were at least three times more active than PrpD with citrate as substrate but their activities with 2-methylcitrate were very low (0·6% of the activity observed with PrpD), and activities with 2-methyl-cis-aconitate also very low (1·0% of the rates obtained with comparable amounts of cis-aconitate). In parallel studies with the YbhJ protein purified to 50% homogeneity from induced cultures of JRG3259(pGS1576), no aconitase activity (<0·001 U per mg protein) was detected with enzymically synthesized citrate and 2-methylcitrate (Table 5
). A weak hydratase activity was observed with 2-methyl-cis-aconitate, but the possibility that this might be due to a contaminating protein cannot be excluded.
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DISCUSSION |
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Amplified and partially purified preparations of the other potential source of AcnC activity, YbhJ, lacked aconitase activity and ybhJ deletion had no significant effect on the AcnC activity of the AcnAB-null strain. The YbhJ sequence clearly indicated that it is a member of the aconitase protein family with 15/20 active-site residues conserved (including the three iron-liganding cysteine residues) and 22% sequence identity to AcnA. It may also be significant that the domain 4 region of YbhJ is remotely related to part of PrpD, but further work will be needed to identify the function of YbhJ.
The 2-methylcitrate dehydratase gene (prpD) is located in the E. coli propionate operon, which is directly related to the prpRBCDE operon of S. typhimurium that has been extensively characterized by Horswill & Escalante-Semerena (1997 , 1999a
, b
, 2001
). In S. typhimurium LT2 the prp genes encode a transcriptional activator and four enzymes of the 2-methylcitrate cycle that permit growth on propionate as a sole carbon and energy source by catalysing its conversion to pyruvate as shown in Fig. 5
. Propionate is an extremely poor substrate for E. coli K-12 but the pathway is induced for the degradation or co-metabolism of propionate (Textor et al., 1997
). Since the present work was completed, Escalante-Semerena and co-workers have established that the PrpD protein of S. typhimurium is a 2-methylcitrate dehydratase and PrpB is a 2-methylisocitrate lyase (Horswill & Escalante-Semerena, 2001
); see Fig. 5
. They also demonstrated that AcnA and AcnB can hydrate 2-methyl-cis-aconitate to 2-methylisocitrate, and have thus been able to reconstruct the conversion of propionate to pyruvate by the 2-methylcitrate pathway in vitro, using purified Prp and Acn enzymes. They further claimed that this route operates in vivo because studies with an acnA and acnB double mutant showed that aconitase enzymes are essential for propionate catabolism. This is an interesting possibility but since it is likely that aconitase is required for growth on pyruvate (as has been demonstrated in E. coli; Gruer et al., 1997b
), aconitase cannot yet be unequivocally assigned to the hydration of 2-methyl-cis-aconitate in vivo.
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Secondly, S. typhimurium H6PrpD was reported to hydrate cis-aconitate at a rate comparable to 2-methylcitrate dehydration (but it failed to hydrate 2-methyl-cis-aconitate to 2-methylisocitrate), citrate was a poor substrate, and isocitrate was not a substrate (Horswill & Escalante-Semerena, 2001 ). In contrast E. coli PrpD was unable to use cis-aconitate (or other substrates having double bonds, excluding 2-methyl-cis-aconitate which was not tested) but citrate and isocitrate were good substrates. The failure to detect hydration of cis-aconitate by E. coli PrpD might reflect a very low affinity for cis-aconitate, and likewise for 2-methyl-cis-aconitate. This could be a desirable feature of an irreversible dehydratase, and if citrate and isocitrate are both converted to cis-aconitate the lack of reversibility would explain why PrpD is not an aconitase. In future it will be important to determine the specificities of both PrpD enzymes for each of the four stereoisomers of 2-methylcitrate and to identify potential inhibitory effects that might stem from the presence of unnatural isomers or their dehydration products. The predominant isomers of 2-methylcitrate generated by mammalian citrate synthase are not identical to the natural isomer produced by the 2-methylcitrate synthases (VanRooyen et al., 1994
; Brock et al., 2000
, 2001
) so the 2-methylcitrate dehydratase activity of PrpD was probably tested under suboptimal conditions with respect to its cognate substrate and the presence of an excess of potentially competing isomers. The latter also applies to the S. typhimurium enzyme, where synthetic substrate containing all four isomers was used (Horswill & Escalante-Semerena, 2001
). This might account for some of the discrepancies between the two reports.
The differences in enzyme substrate specificities between the two species also extended to the aconitases. Compared to the S. typhimurium aconitases, H6AcnA and H10AcnB, which are effective 2-methyl-cis-aconitate hydratases but very poor 2-methylcitrate dehydratases (shown for H6AcnA and assumed for H10AcnB; otherwise PrpD would not be essential for propionate catabolism), the corresponding hydratase and dehydratase activities of the E. coli enzymes, AcnA and AcnB, were extremely low with the methylated substrates (Table 5). But again it should be noted that in both cases the enzymes were tested in the presence of a mixture of potentially inhibitory 2-methylcitrate isomers (VanRooyen et al., 1994
). Nevertheless, these differences could explain why propionate is a good substrate for S. typhimurium but not for E. coli unless pre-adapted. Growth on propionate requires not only that the citric acid and 2-methylcitrate cycles operate simultaneously but also the glyoxylate cycle, which has been shown to be the source of oxaloacetate for propionate utilization rather than the phosphopyruvate carboxylation (Textor et al., 1997
). This provides great opportunities for mutual interference and metabolic derangement which might be more acute in E. coli than in S. typhimurium. It has long been known that operation of the glyoxylate cycle is inhibited by lactate and pyruvate, and by generating pyruvate, propionate utilization might be limited by its inhibitory effect on this essential metabolic cycle. In E. coli the adaptation to propionate might involve subtle alterations of regulatory constraints controlling cofactor availability and metabolic flow, improving the ability of aconitase to convert 2-methyl-cis-aconitate to 2-methylisocitrate, allowing aconitase to function normally in the presence of potentially inhibitory methylated analogues, or derepressing another enzyme that can mediate this conversion. In the latter context it may be significant that partially purified YbhJ exhibited a weak 2-methyl-cis-aconitate hydratase activity that might therefore function in the 2-methylcitrate cycle (Fig. 5
). The very close proximity of the ybhJ gene to the lambda attachment site (att
) also raises the interesting possibility that the recombinant prophages previously shown to amplify a cis-aconitate hydratase upon induction (Wilde et al., 1986
) might have promoted phage-mediated transcription of the flanking ybhJ gene after induction, rather than being
acn phages per se.
Many of the questions concerning enzyme substrate specificities and the potential role of YbhJ in the 2-methylcitrate cycle should be resolved ultimately when pure enzymes can be used with pure substrates and when physiological comparisons are made between strains of E. coli that are unadapted and adapted for growth on propionate. Also, despite the major difference concerning the requirement for ironsulphur clusters, it is clear that the PrpD proteins both of S. typhimurium and E. coli have 2-methylcitrate dehydratase activity, and that PrpD is the source of the activity attributed to AcnC in the AcnAB-null strain of E. coli.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 23 July 2001;
revised 28 August 2001;
accepted 30 August 2001.