Simultaneous Fitting of Absorption Spectra and Their Second Derivatives for an Improved Analysis of Protein Infrared Spectra.

Infrared spectroscopy is a powerful tool in protein science due to its sensitivity to changes in secondary structure or conformation. In order to take advantage of the full power of infrared spectroscopy in structural studies of proteins, complex band contours, such as the amide I band, have to be decomposed into their main component bands, a process referred to as curve fitting. In this paper, we report on an improved curve fitting approach in which absorption spectra and second derivative spectra are fitted simultaneously. Our approach, which we name co-fitting, leads to a more reliable modelling of the experimental data because it uses more spectral information than the standard approach of fitting only the absorption spectrum. It also avoids that the fitting routine becomes trapped in local minima. We have tested the proposed approach using infrared absorption spectra of three mixed α/ß proteins with different degrees of spectral overlap in the amide I region: ribonuclease A, pyruvate kinase, and aconitase.

Expression and properties of the mitochondrial and cytosolic forms of aconitase in maize scutellum.

Aconitase (EC 4.2.1.3) catalyzes the reversible interconversion of citrate, cis-aconitate, and D-isocitrate. It operates in mitochondria and cytosol. We investigated the expression of two aconitase genes (Aco1 and Aco4) and activities of the mitochondrial and cytosolic forms in maize (Zea mays L.) scutellum during germination. Both forms were isolated and purified. The cytosolic form had a higher pH optimum (8.0), twice higher affinity to citrate (K(m) 9.5 mM), and slightly lower affinity to D,L-isocitrate (K(m) 1.7 mM) as compared to the mitochondrial form (optimum pH 7.5, K(m) with citrate 21 mM, and K(m) with isocitrate 1.5 mM). The highest activity of both forms of aconitase was observed on the 4th day of germination; then the activity and expression of the cytosolic form sharply decreased, while the mitochondrial form decreased more slowly. The mitochondrial aconitase was more strongly inhibited by H2O2 (half-inhibition at 35 µM) than the cytosolic form (60 µM). Aconitase activity was not detected in the glyoxysomal fraction beyond the cross-contamination level. It is suggested that the mitochondrial form operates in the tricarboxylic acid cycle, whereas the cytosolic form participates in the reactions of the glyoxylate cycle taking place outside the glyoxysome.

Nucleotide second messenger-mediated regulation of a muralytic enzyme in Streptomyces.

Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.

Enhancement of over expression and chaperone assisted yield of folded recombinant aconitase in Escherichia coli in bioreactor cultures.

A major portion of the over expressed yeast mitochondrial aconitase, a large 82 kDa monomeric TCA cycle enzyme, in Escherichia coli led to the formation of inclusion bodies. Bacterial chaperonin GroEL mediated the correct folding of aconitase with the assistance of its co-chaperonin GroES in an ATP dependent manner. Till date the chaperonin assisted folding of aconitase was limited to the shake flask studies with relatively low yields of folded aconitase. No attempt had yet been made to enhance the yield of chaperone mediated folding of aconitase using a bioreactor. The current report deals with the effect of co-expression of GroEL/GroES in the production of soluble, biologically active recombinant aconitase in E. coli by cultivation in a bioreactor at different temperatures under optimized conditions. It revealed that the yield of functional aconitase was enhanced, either in presence of co-expressed GroEL/ES or at low temperature cultivation. However, the outcome from the chaperone assisted folding of aconitase was more pronounced at lower temperature. A 3-fold enhancement in the yield of functional aconitase from the bioreactor based chaperone assisted folding was obtained as compared to the shake flask study. Hence, the present study provides optimized conditions for increasing the yield of functional aconitase by batch cultivation in a bioreactor.

Iron-dependent RNA-binding activity of Mycobacterium tuberculosis aconitase.

Cellular iron levels are closely monitored by iron regulatory and sensor proteins of Mycobacterium tuberculosis for survival inside macrophages. One such class of proteins systematically studied in eukaryotes and reported in a few prokaryotes are the iron-responsive proteins (IRPs). These IRPs bind to iron-responsive elements (IREs) present at untranslated regions (UTRs) of mRNAs and are responsible for posttranscriptional regulation of the expression of proteins involved in iron homeostasis. Amino acid sequence analysis of M. tuberculosis aconitase (Acn), a tricarboxylic acid (TCA) cycle enzyme, showed the presence of the conserved residues of the IRP class of proteins. We demonstrate that M. tuberculosis Acn is bifunctional. It is a monomeric protein that is enzymatically active in converting isocitrate to cis-aconitate at a broad pH range of 7 to 10 (optimum, pH 8). As evident from gel retardation assays, M. tuberculosis Acn also behaves like an IRP by binding to known mammalian IRE-like sequences and to predicted IRE-like sequences present at the 3' UTR of thioredoxin (trxC) and the 5' UTR of the iron-dependent repressor and activator (ideR) of M. tuberculosis. M. tuberculosis Acn when reactivated with Fe(2+) functions as a TCA cycle enzyme, but upon iron depletion by a specific iron chelator, it behaves like an IRP, binding to the selected IREs in vitro. Since iron is required for the Acn activity and inhibits the RNA-binding activity of Acn, the two activities of M. tuberculosis Acn are mutually exclusive. Our results demonstrate the bifunctional nature of M. tuberculosis Acn, pointing to its likely role in iron homeostasis.

[Catalytic properties of cytoplasmic and mitochondrial aconitate hydratase from rat cardiomyocytes]. / Kataliticheskie svoistva tsitoplazmaticheskoi i mitokhondrial'noi akonitatgidratazy iz kardiomiotsitov krysy.

Enzymatic activity of aconitate hydratase (aconitase, EC 4.2.1.3) from the rat heart is localized in the cytoplasm (65%) and mitochondria (35%). Cytoplasmic and mitochondrial forms of aconitate hydratase were separated by ion-exchange chromatography on CM-Cellulose and CM-Sephadex. The two forms have similar molecular weight, optimal pH range, and spectral properties; however, they have different chromatography properties, Km for citrate and isocitrate, as well as sensitivity to Fe2+ ions.

Sulfolobus aconitase, a regulator of iron metabolism?

The aconitase of Sulfolobus solfataricus, a hyperthermophilic crenarchaeon, was cloned and heterologously expressed in Escherichia coli. Enzymic analyses and EPR measurements indicated clearly that the iron-sulphur cluster of the thermophilic aconitase was already inserted in the mesophilic host. The enzyme was purified to a specific activity of approx. 44 units/mg and to 90% homogeneity. The enzymic parameters of the recombinant aconitase turned out to be in the same range as the respective values for the previously characterized native enzyme from the closely related S. acidocaldarius. Based on its primary sequence, the recombinant aconitase is closely related to bacterial A-like and to eukaryotic iron regulatory protein-like proteins. Specific aconitase activities in cytosolic extracts of S. acidocaldarius were found to be decreased markedly in iron-starved compared with iron-repleted cells. However, no differences in aconitase levels between iron-starved and iron-supplemented cells could be detected by immunostaining.

Induction of aconitate hydratase in hepatocytes of starving rats.

Induction of the activity of aconitate hydratase (AH) was observed in rat hepatocytes under the conditions of food deprivation. The increase in AH activity after 4 days of starvation in the studied tissues was from 0.57 to 2.05 U/g crude liver weight. The induction of aconitase was associated both with the cytoplasmic and mitochondrial AH isoforms. The activities of cytosolic and mitochondrial AH isoforms in starving animals consisted of 83 and 17% of the total activity, respectively. The cytoplasmic and mitochondrial isoforms of the enzyme with specific activities 11.1 and 6.13 U/mg protein, respectively, were obtained by a five-step purification procedure that included fractionation with ammonium sulfate, ion-exchanging chromatography on DEAE-Toyopearl and gel filtration. The purified preparations of these AH isoforms were electrophoretically homogenous. The molecular weights of these isoforms were estimated and several kinetic and regulatory properties were studied.

AcnC of Escherichia coli is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates.

Escherichia coli possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by acnAB double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the prpD gene of the propionate catabolic operon (prpRBCDE) is identical to AcnC. Inactivation of prpD abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of ybhJ, an unidentified acnA paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate cis-aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron-sulphur protein (M(r) 54000) having one unstable [2Fe-2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe(2+) under reducing conditions.

Purification and characterization of the first archaeal aconitase from the thermoacidophilic Sulfolobus acidocaldarius.

The first archaeal aconitase was isolated from the cytosol of the thermoacidophilic Sulfolobus acidocaldarius. Interestingly, the enzyme was copurified with an isocitrate lyase. This enzyme, directly converting isocitrate, the reaction product of the aconitase reaction, was also unknown in crenarchaeota, thus far. Both proteins could only be separated by SDS gel electrophoresis yielding apparent molecular masses of 96 kDa for the aconitase and 46 kDa for the isocitrate lyase. Despite of its high oxygen sensitivity, the aconitase could be enriched 27-fold to a specific activity of approximately 55 micromol x min(-1) x mg(-1), based on the direct aconitase assay system. Maximal enzyme activities were measured at pH 7.4 and the temperature optimum for the archaeal enzyme was recorded at 75 degrees C, slightly under the growth optimum of S. acidocaldarius around 80 degrees C. Thermal inactivation studies of the aconitase revealed the enzymatic activity to be uninfluenced after one hour incubation at 80 degrees C. Even at 95 degrees C, a half-life of approximately 14 min was determined, clearly defining it as a thermostable protein. The apparent K(m) values for the three substrates cis-aconitate, citrate and isocitrate were found as 108 microM, 2.9 mM and 370 microM, respectively. The aconitase reaction was inhibited by the typical inhibitors fluorocitrate, trans-aconitate and tricarballylate. Amino-acid sequencing of three internal peptides of the S. acidocaldarius aconitase revealed the presence of highly conserved residues in the archaeal enzyme. By amino-acid sequence alignments, the S. acidocaldarius sequence was found to be highly homologous to either other putative archaeal or known eukaryal and bacterial sequences. As shown by EPR-spectroscopy, the enzyme hosts an interconvertible [3Fe--4S] cluster.

Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China.

An enzyme analysis of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China was conducted using a horizontal starch gel electrophoresis in order to elucidate their genetic relationships. A total of eight enzymes was employed from two different kinds of buffer systems. Two loci from each enzyme of aconitase and esterase (alpha-Na and beta-Na); and only one locus each from six enzymes, glucose-6-phosphate dehydrogenase (G6PD), alpha-glycerophosphate dehydrogenase (GPD), 3-hydroxybutyrate dehydrogenase (HBDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphoglucomutase (PGM) were detected. Most of loci in two populations of C. sinensis showed homozygous monomorphic banding patterns and one of them, GPD was specific as genetic markers between two different populations. However, esterase (alpha-Na), GPD, HBDH and PGI loci showed polymorphic banding patterns. Two populations of C. sinensis were more closely clustered within the range of genetic identity value of 0.998-1.0. In summarizing the above results, two populations of C. sinensis employed in this study showed mostly monomorphic enzyme protein banding patterns, and genetic differences specific between two populations.

Recognition of a sequestered self peptide by influenza virus-specific CD8+ cytolytic T lymphocytes.

The Ag receptors on CD8+ CTL recognize foreign antigenic peptides associated with cell surface MHC class I molecules. Peptides derived from self proteins are also normally presented by MHC class I molecules. Here we report that an H-2Kd-restricted murine CD8+ CTL clone directed to an influenza hemagglutinin epitope can recognize a peptide derived from the murine mitochondrial aconitase enzyme in association with H-2Kd molecules. Surprisingly, this self peptide is not normally displayed on the cell surface associated with the restricting MHC class I molecule. Several lines of evidence suggest that this self peptide, although requiring association with the Kd molecule for CTL recognition, is not associated with this or other MHC class I allele under physiologic conditions in intact cells. Rather, it is sequestered in the cytoplasm associated with a carrier protein and is released only upon cell disruption. These results suggest a means of restricting the entry of self peptide into the class I pathway. In addition, this finding raises the possibility that self peptides sequestered within the cell can, after release from damaged cells, interact with MHC class I molecules on bystander cells and trigger autoimmune injury by virus-specific CTLs during viral infection.

Identification of fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry.

Iron plays a critical role in the pathophysiology of Mycobacterium tuberculosis. To gain a better understanding of iron regulation by this organism, we have used two-dimensional (2-D) gel electrophoresis, mass spectrometry, and database searching to study protein expression in M. tuberculosis under conditions of high and low iron concentration. Proteins in cellular extracts from M. tuberculosis Erdman strain grown under low-iron (1 microM) and high-iron (70 microM) conditions were separated by 2-D polyacrylamide gel electrophoresis, which allowed high-resolution separation of several hundred proteins, as visualized by Coomassie staining. The expression of at least 15 proteins was induced, and the expression of at least 12 proteins was decreased under low-iron conditions. In-gel trypsin digestion was performed on these differentially expressed proteins, and the digestion mixtures were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Partial sequence data on some of the peptides were obtained by using after source decay and/or collision-induced dissociation. The fragmentation data were used to search computerized peptide mass and protein sequence databases for known proteins. Ten iron-regulated proteins were identified, including Fur and aconitase proteins, both of which are known to be regulated by iron in other bacterial systems. Our study shows that, where large protein sequence databases are available from genomic studies, the combined use of 2-D gel electrophoresis, mass spectrometry, and database searching to analyze proteins expressed under defined environmental conditions is a powerful tool for identifying expressed proteins and their physiologic relevance.

The second aconitase (AcnB) of Escherichia coli.

The second aconitase (AcnB) of Escherichia coli was partially purified from an acnA::kanR mutant lacking AcnA, and the corresponding polypeptide identified by activity staining and weak cross-reactivity with AcnA antiserum. The acnB gene was located at 2 center dot 85 min (131 center dot 6 kb) in a region of the chromosome previously assigned to two unidentified ORFs. Aconitase specific activities were amplified up to fivefold by infection with lambdaacnB phages from the Kohara lambda-E. coli gene library, and up to 120-fold (50% of soluble protein) by inducing transformants containing a plasmid (pGS783) in which the acnB coding region is expressed from a regulated T7 promoter. The AcnB protein was purified to > or = 98% homogeneity from a genetically enriched source (JRG3171) and shown to be a monomeric protein of Mr 100 000 (SDS-PAGE) and 105 000 (gel filtration analysis) compared with Mr 93 500 predicted from the nucleotide sequence. The sequence identity between AcnA and AcnB is only 17% and the domain organization of AcnA and related proteins (1-2-3-linker-4) is rearranged in AcnB (4-1-2-3).

Identification of a multienzyme complex of the tricarboxylic acid cycle enzymes containing citrate synthase isoenzymes from Pseudomonas aeruginosa.

A multienzyme complex of tricarboxylic acid cycle enzymes, catalysing the consecutive reactions from fumarate to 2-oxoglutarate, has been identified in extracts of Pseudomonas aeruginosa prepared by gentle osmotic lysis of the cells. The individual enzyme activities of fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase can be used to reconstitute the complex. The citrate synthase isoenzymes, CSI and CSII, from this organism can be used either together or as the individual activities to reconstitute the complex. No complex can be reformed in the absence of CSI or CSII. Which CS isoenzyme predominates in the complex depends on the phase of growth at which the cells were harvested and the extract prepared. More CSI was found in the complex during exponential growth, whereas CSII predominated during the stationary phase. The results support the idea of a 'metabolon' in this organism, with the composition of the CS component varying during the growth cycle.

Spectroscopic characterisation of an aconitase (AcnA) of Escherichia coli.

A spectroscopic study of an aconitase, AcnA, from Escherichia coli is presented. The amino acid sequence of AcnA has 53% identity with mammalian cytosolic aconitase (c-aconitase) which is the translational regulator known as iron regulatory factor (IRF). In the [3Fe-4S](+)-containing, inactive state, AcnA displays an EPR signal which is not unlike the corresponding signal from mammalian mitochondrial aconitase (m-aconitase) but is even more similar to the signal from c-aconitase. This is perhaps related to the greater similarity of the AcnA amino acid sequence with c-aconitase. Magnetic circular dichroism (MCD) spectroscopy has revealed that the electronic structure of the [3Fe-4S] cluster of AcnA must be similar to, but not identical to that of m-aconitase. Whilst the [Fe-4S] clusters from both of these enzymes display some features in their MCD spectra common to [3Fe-4S] clusters in general, their spectra overall are unique and indicate that the Fea atom of the [4Fe-4S] form is not the only unusual feature of the [Fe-S] clusters of aconitases. Active [4Fe-4S]-containing AcnA can be reduced to yield an EPR signal due to a [4Fe-4S]+ cluster which is indistinguishable from the signals from the [4Fe-4S]+ cluster in the mammalian enzymes. However, in contrast to the mammalian enzymes, the EPR signals of the cluster in AcnA are not significantly perturbed upon the addition of substrate. Furthermore, the catalytic activity of [Fe-4S](2+)-containing AcnA is fivefold higher than that of m-aconitase. The mechanistic implications of these data are discussed. A novel S = 1/2 EPR signal with g approximately 2 was observed in AcnA upon treatment with EDTA. The species giving rise to this signal is proposed to be an intermediate in cluster deconstruction.

Structure, genomic organization, and expression of the Arabidopsis thaliana aconitase gene. Plant aconitase show significant homology with mammalian iron-responsive element-binding protein.

We report the purification of the unstable aconitase enzyme from melon seeds and the NH2-terminal amino acid sequence determination. Antibodies raised against this protein enabled the first isolation and characterization of cDNA encoding aconitase in plants. A full-length cDNA clone of 3210 base pairs was isolated from a library of cDNA clones derived from immature pods of Arabidopsis thaliana. The amino acid sequence deduced from the open reading frame includes the sequence obtained by direct sequencing of the NH2 terminus of the purified enzyme. Genomic clones of the aconitase gene were isolated, and comparison of the cDNA and genomic sequences reveals that the coding sequence is divided among 20 exons. There are five putative sites for transcription initiation. The aconitase gene is constitutively expressed, but at a low level, during most developmental stages, with a dramatic increase during seed and pollen maturation and during germination. Surprisingly, plant aconitases have reasonably high homology to binding proteins for iron-responsive elements from mammalian species, opening the possibility that a similar type of translational regulation occurs in plants.

Aconitase is a sensitive and critical target of oxygen poisoning in cultured mammalian cells and in rat lungs.

The effect of hyperoxia on activity of the superoxide-sensitive citric acid cycle enzyme aconitase was measured in cultured human epithelial-like A549 cells and in rat lungs. Rapid and progressive loss of > 80% of the aconitase activity in A549 cells was seen during a 24-hr exposure to a PO2 of 600 mmHg (1 mmHg = 133 Pa). Inhibition of mitochondrial respiratory capacity correlated with loss of aconitase activity in A549 cells exposed to hyperoxia, and this effect could be mimicked by fluoroacetate (or fluorocitrate), a metabolic poison of aconitase. Exposure of rats to an atmospheric PO2 of 760 mmHg or 635 mmHg for 24 hr caused respective 73% and 61% decreases in total lung aconitase activity. We propose that early inactivation of aconitase and inhibition of the energy-producing and biosynthetic reactions of the citric acid cycle contribute to the sequelae of lung damage and edema seen during exposure to hyperoxia.

Lack of aconitase in glyoxysomes and peroxisomes.

The aim of this work was to find out whether aconitase [citrate (isocitrate) hydro-lyase, EC 4.2.1.3] which is rapidly inactivated by H2O2, is present in the microbodies from plant cells. The separation of intact organelles from castor-bean (Ricinus communis) endosperm and potato (Solanum tuberosum) tuber indicated that aconitase activity is essentially limited to the mitochondria and cytosol fraction, but was not detected in highly purified castor-bean endosperm and potato tuber peroxisomes. An isotropic e.p.r. signal of the type expected for the 3Fe cluster of oxidized aconitase was not detected in microbodies. In immunoblot analyses, antibodies raised against potato tuber mitochondrial aconitase did not cross-react with any glyoxysomal or peroxisomal protein. Positive reactions were found for cytosol fraction and mitochondria of castor-bean endosperm. The operation of the full glyoxylate cycle in isolated glyoxysomes requires the presence of aconitase in the incubation medium. It is concluded that glyoxysomes are probably devoid of aconitase and that the glyoxylate cycle requires a detour via the cytosol, which contains a powerful aconitase activity.

Purification and characterization of cytosolic aconitase from beef liver and its relationship to the iron-responsive element binding protein.

In recent reports attention has been drawn to the extensive amino acid homology between pig heart, yeast, and Escherichia coli aconitases (EC 4.2.1.3) and the iron-responsive element binding protein (IRE-BP) of mammalian cells [Rouault, T. A., Stout, C. D., Kaptain, S., Harford, J. B. & Klausner, R. D. (1991) Cell 64, 881-883.; Hentze, M. W. & Argos, P. (1991) Nucleic Acids Res. 19, 1739-1740.; Prodromou, C., Artymiuk, P. J. & Guest, J. R. (1992) Eur. J. Biochem. 204, 599-609]. Iron-responsive elements (IREs) are stem-loop structures located in the untranslated regions of mRNAs. IRE-BP is required in the posttranscriptional regulation of ferritin mRNA translation and stabilization of transferrin receptor mRNA. In spite of substantial homology between the amino acid sequences of mammalian mitochondrial aconitase and IRE-BP, the mitochondrial protein does not bind IREs. However, there is a second aconitase, found only in the cytosol of mammalian tissues, that might serve as an IRE-BP. To test this possibility, we have prepared sufficient quantities of the heretofore poorly characterized beef liver cytosolic aconitase. This enzyme is isolated largely in its active [4Fe-4S] form and has a turnover number similar to that of mitochondrial aconitase. The EPR spectra of the two enzymes are markedly different. The amino acid composition, molecular weight, isoelectric point, and the sequences of six random peptides clearly show that these physicochemical and structural characteristics are identical to those of IRE-BP, and that c-aconitase is distinctly different from m-aconitase. In addition, both cytosolic aconitase and IRE-BP can have aconitase activity or function as IRE-BPs, as shown in the following paper and elsewhere [Zheng, L. Kennedy, M. C., Blondin, G. A., Beinert, H. & Zalkin, H. (1992) Arch. Biochem. Biophys., in press]. This leads us to the conclusion that cytosolic aconitase is IRE-BP.