Aluminum Triggers Decreased Aconitase Activity via Fe-S Cluster Disruption and the Overexpression of Isocitrate Dehydrogenase and Isocitrate Lyase

Although aluminum is known to be toxic to most organisms, its precise biochemical interactions are not fully understood. In the present study, we demonstrate that aluminum promotes the inhibition of aconitase (Acn) activity via the perturbation of the Fe-S cluster in Pseudomonas fluorescens. Despite the significant decrease in citrate isomerization activity, cellular survival is assured by the overexpression of isocitrate lyase and isocitrate dehydrogenase (IDH)-NADP+. 13C NMR spectroscopic studies, Blue Native PAGE, and Western blot analyses indicated that although the decrease in Acn activity is concomitant with the increase of aluminum in the culture, the amount of Acn expressed is not sensitive to the concentration of the trivalent metal. A 6-fold decrease in Acn activity and no discernable change in protein content in aluminum-stressed cultures were observed. The addition of Fe(NH4)2(SO4)2 in a reducing environment led to a significant recovery in Acn activity. This enzymatic activity reverted to normal levels when aluminum-stressed cells were transferred to either a control or an iron-supplemented medium. The overexpression of the two isocitrate-metabolizing enzymes isocitrate lyase and IDH-NADP+ appears to mitigate the deficit in Acn activity. The levels of these enzymes are dependent on the aluminum content of the culture and appear to be under transcriptional control. Hence, the regulation of the enzymes involved in the homeostasis of isocitrate constitutes a pivotal component of the global metabolic strategy that ensures the survival of this organism in an aluminum citrate environment. Although aluminum is known to be toxic to most organisms, its precise biochemical interactions are not fully understood. In the present study, we demonstrate that aluminum promotes the inhibition of aconitase (Acn) activity via the perturbation of the Fe-S cluster in Pseudomonas fluorescens. Despite the significant decrease in citrate isomerization activity, cellular survival is assured by the overexpression of isocitrate lyase and isocitrate dehydrogenase (IDH)-NADP+. 13C NMR spectroscopic studies, Blue Native PAGE, and Western blot analyses indicated that although the decrease in Acn activity is concomitant with the increase of aluminum in the culture, the amount of Acn expressed is not sensitive to the concentration of the trivalent metal. A 6-fold decrease in Acn activity and no discernable change in protein content in aluminum-stressed cultures were observed. The addition of Fe(NH4)2(SO4)2 in a reducing environment led to a significant recovery in Acn activity. This enzymatic activity reverted to normal levels when aluminum-stressed cells were transferred to either a control or an iron-supplemented medium. The overexpression of the two isocitrate-metabolizing enzymes isocitrate lyase and IDH-NADP+ appears to mitigate the deficit in Acn activity. The levels of these enzymes are dependent on the aluminum content of the culture and appear to be under transcriptional control. Hence, the regulation of the enzymes involved in the homeostasis of isocitrate constitutes a pivotal component of the global metabolic strategy that ensures the survival of this organism in an aluminum citrate environment. Metabolism is the foundation of all living organisms, and any biological function is the manifestation of the global cellular metabolism. Hence, any cellular behavior is a direct or indirect product of its metabolism. The enzymes/metabolites participating in metabolism provide a precise snapshot of a cellular phenotype (1Sanford K. Soucaille P. Whited G. Chotani G. Curr. Opin. Microbiol. 2002; 5: 318-322Crossref PubMed Scopus (52) Google Scholar, 2Goodacre R. Drug Discov. Today. 2004; 9: 260-261Crossref PubMed Scopus (15) Google Scholar). As part of our study on molecular adaptation, we have uncovered an interesting model system that allows deciphering the metabolic reconfiguration evoked by metal stress. The metal toxicant was supplied to the microbe Pseudomonas fluorescens, complexed to citrate, the only carbon source. The role of oxalate and phosphatidylethanolamine in the immobilization of aluminum has been demonstrated recently (3Hamel R. Appanna V.D. Biochim. Biophys. Acta. 2003; 1619: 70-76Crossref PubMed Scopus (29) Google Scholar, 4Hamel R.D. Appanna V.D. J. Inorg. Biochem. 2001; 87: 1-8Crossref PubMed Scopus (35) Google Scholar). It appears that the cellular metabolism is reconfigured with the aim of providing the metabolic precursors that allow for the survival of the organism in an aluminum environment. Hence, an aluminum-adapted phenotype with an entirely different set of metabolic pathways than in the wild type is promoted. Citrate, the sole carbon source utilized in this system, is known to be cleaved in various organisms, primarily by the enzymes citrate-lyase (CL), 1The abbreviations used are: CL, citrate-lyase; Acn, aconitase; IDH, isocitrate dehydrogenase; ICL, isocytrate lyase; DTT, dithiothreitol; CFE, cell-free extract; BN, Blue Native; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,-1-bis(hydroxymethyl)ethyl]glycine. 1The abbreviations used are: CL, citrate-lyase; Acn, aconitase; IDH, isocitrate dehydrogenase; ICL, isocytrate lyase; DTT, dithiothreitol; CFE, cell-free extract; BN, Blue Native; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; Tricine, N-[2-hydroxy-1,-1-bis(hydroxymethyl)ethyl]glycine. ATP-citrate-lyase (ATP-CL), and Acn. Whereas CL mediates the cleavage of citrate to acetate and oxaloacetate, ATP-CL catalyzes the degradation of tricarboxylic acid into acetyl-CoA and oxaloacetate (5Schneider K. Dimroth P. Bott M. FEBS Lett. 2000; 483: 165-168Crossref PubMed Scopus (23) Google Scholar, 6Adams I.P. Dack S. Dickinson F.M. Ratledge C. Biochim. Biophys. Acta. 2002; 1597: 36-41Crossref PubMed Scopus (20) Google Scholar). The latter is also referred to as a lipogenic enzyme, because it participates in the generation of acetyl-CoA, a key precursor in the biosynthesis of fatty acid, and is regulated via the phosphorylation/dephosphorylation of its histidine residues (7Elshourbagy N.A. Near J.C. Kmetz P.J. Sathe G.M. Southan C. Strickler J.E. Gross M. Young J.F. Wells T.N. Groot P.H. J. Biol. Chem. 1990; 265: 1430-1435Abstract Full Text PDF PubMed Google Scholar, 8Tuhackova Z. Krivanek J. Biochem. Biophys. Res. Commun. 1996; 218: 61-66Crossref PubMed Scopus (12) Google Scholar). CL, on the other hand, is usually invoked by microorganisms utilizing citrate in anoxic environments (9Clark D.P. FEMS Microbiol. Lett. 1990; 55: 245-249Crossref PubMed Google Scholar). Aconitases, the other group of enzymes that are involved in the metabolism of citrate, are Fe-S proteins having a predominantly [4Fe-4S] cluster in the enzymatically active form. They catalyze the reversible isomerization of citrate to isocitrate, a key step in the tricarboxylic acid cycle. The isocitrate is metabolized subsequently by IDH-NAD+ to give α-ketoglutarate and NADH. Acn is sensitive to the oxygen gradient and the cellular iron status (10Fridovich I. Annu. Rev. Biochem. 1995; 64: 97-112Crossref PubMed Scopus (2704) Google Scholar). These two factors play an important role in the reactivity of Acn. In mammalian systems, Acn with a [3Fe-4S] cluster serves as a regulatory protein that controls the stability and translation of mRNAs encoding proteins involved in iron and energy homeostasis (11Beinert H. Kennedy M.C. Stout C.D. Chem. Rev. 1996; 96: 2335-2374Crossref PubMed Scopus (470) Google Scholar, 12Haile D.J. Rouault T.A. Harford J.B. Kennedy M.C. Blondin G.A. Beinert H. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11735-11739Crossref PubMed Scopus (264) Google Scholar). The regulatory Acn, referred to as iron-responsive proteins, binds to the iron-responsive elements localized in the RNA-stem-loop. Aluminum is the most abundant metal in the environment and is known to be toxic to all organisms. It may substitute for such metals as iron and calcium and consequently help destabilize biological activity. There are accumulating reports (13Nayak P. Environ. Res. 2002; 89: 101-115Crossref PubMed Scopus (439) Google Scholar) that suggest that aluminum interferes with iron homeostasis and severely impedes cellular metabolism. Aluminum also is known to favor the generation of an oxidative environment because of its ability to create a labile iron pool and to interact with membrane lipids (14Exley C. Free Radic. Biol. Med. 2004; 36: 380-387Crossref PubMed Scopus (335) Google Scholar). Although most organisms succumb to the toxic influence of aluminum, some living systems are known to elaborate intricate strategies to fend off the dangers associated with an aluminum-rich environment (15Appanna V.D. Hamel R.D. Recent Res. Dev. Microbiol. 1999; 3: 615-663Google Scholar, 16Ma J.F. Zheng S.J. Matsumoto H. Hiradale S. Nature. 1997; 390: 559-560Crossref PubMed Scopus (46) Google Scholar). We have shown the ability of P. fluorescens to tolerate millimolar amounts of aluminum complexed to citrate, the only carbon source (17Hamel R. Levasseur R. Appanna V.D. J. Inorg. Biochem. 1999; 76: 99-104Crossref PubMed Scopus (47) Google Scholar). The present study was aimed at elucidating the enzymes involved in the metabolism of citrate in the presence of aluminum. Here we have shown that aluminum severely inhibits Acn activity by perturbing the Fe-S cluster and that the degradation of citrate is aided by two downstream enzymes, ICL and IDH-NADP+. The overexpression of ICL and IDH contributed effectively in the metabolism of the tricarboxylic acid even under markedly diminished Acn activity. It appears that the rapid cleavage of isocitrate, the product generated by Acn, via ICL and IDH provided an efficient route for the survival of this organism in an aluminum citrate environment. The significance of this aluminum-evoked metabolic shift is discussed, and the pivotal role of a metabolic circuit operative in this organism is explained. Bacterial Culture and Cell-free Extracts—P. fluorescens, ATCC 13525, was maintained (on 2% agar) and grown in a mineral medium consisting of Na2HPO4 (6.0 g), KH2PO4 (3.0 g), NH4Cl (0.8 g), MgSO4 (0.2 g), and citric acid (4.0 g/liter deionized distilled water). Trace elements (FeCl3·6H2O(2 μm), MgCl2·4H2O(1 μm), Zn(NO3)2·6H2O (0.05 μm), CaCl2 (1 μm), CoSO4·7H2O (0.25 μm), CuCl2·2H2O (0.1 μm), and NaMoO4·2H2O (0.1 μm)) were also added. In the aluminum-stressed medium, citric acid was complexed with AlCl3 in a ratio of 19 mm citrate to 15 mm AlCl3. The pH was adjusted to 6.8 with dilute NaOH and 200-ml amounts of media were dispensed in 500-ml Erlenmeyer were with of cells grown in a medium with the metal and on a model at other metals were the citrate concentration was also 19 and and iron were present at and mm In the 19 mm was were by for at and with and with mm mm DTT, and mm were in the mm acid, a at for 15 at The cells were the cell-free by at for at The of the was and at for at to membrane and The was at for to a were on in the or at for to a of were by the as a Biochem. PubMed Scopus Google Scholar). of protein were at in a mm mm DTT, mm mm citrate or metal citrate and mm Acn activity was by the of at J. Biol. Chem. Full Text PDF PubMed Google Scholar). ICL activity was by with the of Biochem. 1999; PubMed Scopus Google Scholar). of protein and mm isocitrate were used in the was utilized as the IDH-NADP+ was in a mm mm mm isocitrate, mm and mm activity was by the of at for and by the α-ketoglutarate with was used as the the of Acn and ICL, the were with citrate, and the of was to the of Acn, ICL, and IDH-NADP+ the of and was 13C NMR NMR analyses were the at for were in mm mm with of protein mm acid or The were in for and were by the in a for were by at for of the were with of were for and were to Blue Native PAGE, Western and Native was to the of H. G. Biochem. PubMed Scopus Google Scholar). The system was and with a gradient were to the protein were in mm BisTris, mm acid and of was into were under was used for the and was to when the proteins the The mm Tricine, 15 mm BisTris, Blue at was to the mm Tricine, 15 mm at the the was the was of the The were subsequently in an mm mm for 15 Acn activity was by the of via and an consisting of mm citrate, mm IDH and ICL was with the and were the This was also by for generated the cleavage of dehydrogenase was utilized as the Biochim. Biophys. Acta. PubMed Scopus Google Scholar). IDH activity was with isocitrate and and was under the as content was by Blue and Western a system was to the of Nature. PubMed Scopus Google Scholar). A and were were in mm 2% and 2% at for was at a and were and to were on and as as the of or the was in protein mm mm for membrane was utilized for The were by the with in mm for The were with the for or aconitase provided by R. of and ICL K. The of The of the proteins was with the system a at the were with was Aluminum and of Acn, IDH, and were grown and as were at various and the were The and the protein content of Acn, ICL, and IDH were by and Western the influence of aluminum on these enzymes, cells were in media with and of aluminum. The and of the enzymes were of and of protein of aluminum-stressed cells were transferred to various citrate media with 15 mm and were with control cells transferred to aluminum medium. an of cells were and the cellular were for enzymatic and protein In of Acn and of Fe-S of Acn was with of protein of in the presence of mm and mm Fe(NH4)2(SO4)2 in the The was for and Acn activity was The of the Fe-S cluster was by protein of cultures with of aluminum with an In aluminum cultures transferred to the control medium was also The Fe-S cluster at was C. C. T.A. J.C. 2003; PubMed Scopus Google Scholar, C. M. M.C. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus (52) Google Scholar). of Aluminum on Acn and demonstrate that in the presence of aluminum the ability of Acn to citrate to isocitrate was that when cells grown in media with the metal was with acid for Acn was to citrate to This was indicated by the presence of the present in a at in NMR Scholar). when cells grown in the presence of aluminum was the at was that the presence of aluminum in the culture a on Acn activity not the was or in the presence of the tricarboxylic acid was metabolized of Acn on and was used to this decrease in Acn activity. The or of in the aluminum-stressed cells indicated that this was severely in these cultures the of Acn activity in the aluminum-stressed cells was by a of protein Western blot analyses were and The protein levels of Acn were to be not in aluminum-stressed that the change in activity was not because of protein of aluminum on activity and of Acn. and of Acn on and are activity of activity Acn in and P. fluorescens, of of and Aluminum activity was at different by activity on significant change in Acn activity was of in cells in control medium. The aluminum-stressed cells a decrease in Acn activity to of with the immobilization of aluminum as a and (3Hamel R. Appanna V.D. Biochim. Biophys. Acta. 2003; 1619: 70-76Crossref PubMed Scopus (29) Google a increase in Acn activity was observed. The in Acn activity was in cells to mm grown with mm no decrease in Acn activity. a concentration of mm was a change in Acn activity. the presence of iron to have a on this These were by the of at not Acn in with and the decrease in Acn activity was to the toxic influence of aluminum, the cells were grown in various media different metals complexed to citrate and as the sole carbon As shown by NMR and activity Acn activity in aluminum-stressed cells was to a 6-fold decrease of Acn activity in the aluminum-stressed cells when known was complexed to citrate, a decrease in Acn activity was observed. when complexed to citrate was the was no significant perturbation in Acn activity. In (1 the under was the only a in Acn activity was when was utilized as the source of a decrease in Acn activity was with the control citrate medium. of and the that aluminum was the in a decrease in Acn activity, cells were to aluminum and transferred to various citrate media aluminum, and The cells were subsequently for The aluminum-stressed cells an increase in Acn activity in the cultures citrate and citrate with in the cultures with no significant in Acn activity was the of Acn to oxidative stress. Although the amount of protein to Acn was not in the presence of either or the enzymatic activity was the of Acn was and Acn activity in were to the significance of the Fe-S cluster in Acn activity in aluminum-stressed Acn activity was the of the with Fe(NH4)2(SO4)2 and DTT, was an increase of at in aluminum-stressed significant change was in the control cells not The with Fe-S cluster perturbation The and that has been shown to be of [4Fe-4S] C. C. T.A. J.C. 2003; PubMed Scopus Google Scholar, C. M. M.C. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus (52) Google Scholar) was markedly different in control than in aluminum-stressed In the the was in the a significant in the was The of this with concentration of aluminum in the medium. in aluminum-stressed cells transferred to a medium of the the of Aluminum on the and of IDH and ICL in P. an involved in the of isocitrate to α-ketoglutarate with concomitant of a activity in the the aluminum-stressed cells with the control Blue of IDH on demonstrated levels of protein associated with IDH in the aluminum-stressed cells with the control cells A and an of IDH also was in the aluminum-stressed The cells grown in the control medium were transferred into media with aluminum, aluminum with and aluminum with In the aluminum medium, a increase was of in the aluminum media with and no significant increase was ICL, known to isocitrate product of to and was also to a increase in the aluminum-stressed The of ICL to aluminum and its overexpression in the aluminum-stressed cells were also demonstrated by and Western blot analyses of aluminum on ICL activity and protein activity of for ICL on is for ICL cells aluminum citrate cells for in media of aluminum cells in aluminum citrate media cells in aluminum citrate media cells in aluminum citrate media of protein were of to and by the and P. was with citrate, and the of was As in the amounts of by control and aluminum-stressed were even Acn activity in the aluminum-stressed cultures was severely the and α-ketoglutarate generated by the control and aluminum-stressed not appear to The of ICL and IDH to for the of Acn activity in the cells to aluminum of citrate to and α-ketoglutarate in P. mm was utilized as an of mm was utilized as an of ICL, and mm was utilized as an of in a The the pivotal role that ICL and IDH in the of P. fluorescens when the organism was to aluminum as a toxicant and citrate as the sole carbon source. The influence of aluminum on the Acn in was also The decrease in Acn activity was sensitive to the concentration of aluminum in the medium and was not by a decrease in protein by the perturbation of the Fe-S Hence, Acn was a of the toxic or by aluminum, with metabolic survival was assured not by an by the overexpression of ICL and IDH, two enzymes downstream of Acn. The of any discernable amounts of citrate-lyase or citrate-lyase indicated that these enzymes are not involved in the of citrate R. and in Pseudomonas fluorescens to Aluminum A of of and of 2003; Google Scholar). In this Acn to be the participating in the of citrate, even it is a of aluminum The ability of aluminum to iron and oxygen an environment for Acn to as an (14Exley C. Free Radic. Biol. Med. 2004; 36: 380-387Crossref PubMed Scopus (335) Google Scholar). This oxidative and by aluminum an Acn with than activity. it has been G. S. A. G. Free Radic. Biol. Med. 2002; PubMed Scopus Google Scholar, Annu. Rev. 2000; PubMed Scopus Google Scholar) that the of Acn to these this protein to iron and oxygen and allows it to the homeostasis of iron and In the present study, the of Acn in the control and aluminum-stressed cultures were the were markedly The Acn the aluminum-stressed diminished activity. This activity was on with and Fe(NH4)2(SO4)2 or when the aluminum-stressed cells were to an medium. It is important to that the of activity in the aluminum-stressed culture was to the in the medium as the sole source of Hence, it is that although the activity was diminished in it was to the of Acn in the citrate cultures with in the medium with In the presence of and has been It has been that is sensitive to oxidative and participates in the tricarboxylic acid the of is expressed the PubMed Scopus Google Scholar, S. J. 2003; PubMed Scopus Google Scholar). In the present study, and Western blot analyses only to Acn activity. activity was discernable in any other cellular It is that although Acn is sensitive to the presence of or in the it is not by a metal such as Hence, an oxidative environment and a of iron by aluminum be the Acn activity. It is also to that Acn, in this may also be as a involved in the oxygen gradient and iron pool in a role has been demonstrated recently C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). Acn, with a markedly activity, is the key citrate cellular metabolism in P. fluorescens to aluminum citrate, this metabolic strategy may be it is not that other may be to the of the microbe in this aluminum citrate environment. The of the microbe in aluminum medium to the of other enzymes in this V.D. J. M. 1995; Scopus (12) Google Scholar). the two enzymes ICL and IDH-NADP+ that isocitrate, the product of the isomerization of citrate by Acn, appear to be an part of the biochemical strategy invoked by P. fluorescens to this toxic ICL activity that because of the overexpression of this protein is to the presence of aluminum in the medium R. Appanna V.D. S. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google Scholar). The rapid of isocitrate favor the of citrate to The activity of ICL may also help a pivotal to the of aluminum. is to acid, a that is involved in the of the trivalent metal (3Hamel R. Appanna V.D. Biochim. Biophys. Acta. 2003; 1619: 70-76Crossref PubMed Scopus (29) Google Scholar). Hence, it is not that the of ICL may be the role of citrate Acn activity and providing the key for the of This acid has also been to as an Biochim. Biophys. Acta. 2002; PubMed Scopus Google a that may be in the aluminum-stressed by the of isocitrate its cleavage into and the of ICL may favor the of the ability of the control and aluminum-stressed cells to amounts of citrate to such a in also isocitrate as a a increase in aluminum-stressed This activity have a on the of citrate to The of isocitrate the isomerization of citrate to This allow for the degradation of citrate even the activity of Acn is diminished markedly because of the oxidative and environment by aluminum stress. The diminished of Acn activity in aluminum-stressed cells to be by the of the enzymes involved in the degradation of The overexpression of ICL and IDH in this may be such a Hence, a metabolic that the of to be for biological may be an important strategy that living systems to to and cellular The the of enzymes to citrate in an oxidative and environment the to a diminished Acn as the of citrate it is not to that this Acn with perturbation in its Fe-S cluster may be as a regulatory and a diminished enzymatic This metabolic circuit Acn, IDH, and ICL allows for the degradation of citrate and the precursors that under aluminum stress. and α-ketoglutarate may to the oxidative generated by aluminum stress. In a product of ICL, the of acid, a involved in aluminum it is not that the a metabolic reconfiguration in an to with the by aluminum stress. are the of biological and allow the of the biochemical status of an They provide an into the metabolic and the proteins for and Hence, Acn, ICL, IDH, isocitrate, citrate, and α-ketoglutarate are as part of the global metabolic in aluminum-stressed P. fluorescens. In these demonstrate that Acn, a key in cellular energy is a of aluminum The Acn activity is not by a decrease in protein concentration by the perturbation of the Fe-S This the that the tricarboxylic acid may be under the toxic influence of aluminum. In this the survival of the microbe in the aluminum-stressed environment is by the of ICL and IDH, two enzymes that usually downstream to Acn. The of these two enzymes are concomitant with the increase in the protein The of isocitrate via ICL and IDH the degradation of citrate even under diminished Acn This a of enzymes may provide an for the survival of this aluminum-stressed organism and the of metabolic pathways in proteins aimed at a and help provide a snapshot of the and an of the global metabolic operative in P. fluorescens with aluminum. We R. of and K. for for Acn and ICL,

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.