The aconitase of Escherichia coli: purification of the enzyme and molecular cloning and map location of the gene (acn)

The aconitase of Escherichia coli was purified to homogeneity, albeit in low yield (0.6%). It was shown to be a monomeric protein of Mr 95,000 or 97,500 by gel filtration and SDS-PAGE analysis, respectively. The N-terminal amino acid sequence resembled that of the Bacillus subtilis enzyme (citB product), but the similarity at the DNA level was insufficient to allow detection of the E. coli acn gene using a 456 bp citB probe. Phages containing the acn gene were isolated from a lambda-E. coli gene bank by immunoscreening with an antiserum raised against purified bacterial enzyme. The acn gene was located at 28 min (1350 kb) in the physical map of the E. coli chromosome by probing Southern blots with a fragment of the gene. Attempts to locate the gene using the same procedure with oligonucleotide probes encoding segments of the N-terminal amino acid sequence were complicated by the lack of probe specificity and an inaccuracy in the physical map of Kohara et al. (Cell 50, 495-508, 1987). Aconitase specific activity was amplified some 20-200-fold in cultures transformed with pGS447, a derivative of pUC119 containing the acn gene, and an apparent four-fold activation-deactivation of the phagemid-encoded enzyme was observed in late exponential phase. The aconitase antiserum cross-reacted with both the porcine and Salmonella typhimurium (Mr 120,000) enzymes.     

Nitric oxide modulates the activity of tobacco aconitase

Recent evidence suggests an important role for nitric oxide (NO) signaling in plant-pathogen interactions. Additional elucidation of the role of NO in plants will require identification of NO targets. Since aconitases are major NO targets in animals, we examined the effect of NO on tobacco (Nicotiana tabacum) aconitase. The tobacco aconitases, like their animal counterparts, were inhibited by NO donors. The cytosolic aconitase in animals, in addition to being a key redox and NO sensor, is converted by NO into an mRNA binding protein (IRP, or iron-regulatory protein) that regulates iron homeostasis. A tobacco cytosolic aconitase gene (NtACO1) whose deduced amino acid sequence shared 61% identity and 76% similarity with the human IRP-1 was cloned. Furthermore, residues involved in mRNA binding by IRP-1 were conserved in NtACO1. These results reveal additional similarities between the NO signaling mechanisms used by plants and animals.     

Homology between IRE-BP, a regulatory RNA-binding protein, aconitase, and isopropylmalate isomerase.

Iron-responsive elements (IREs) are regulatory RNA elements which serve as specific binding sites for the IRE-binding protein (IRE-BP). Interaction between IREs and IRE-BP induces repression of ferritin mRNA translation and transferrin receptor mRNA stabilization. We describe the identification of extensive amino acid sequence homology between IRE-BP and two known isomerases, aconitase and isopropylmalate (IPM) isomerase. We discuss the implications of this observation with regard to structure/function relationships of IRE-BP. The structural conservation between a regulatory RNA-binding protein and two enzymes involved in intermediary metabolism provides a surprising example of the functional flexibility in biological structures.     

The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein.

Legionella pneumophila has high iron requirements, and its intracellular growth in human monocytes is dependent on the availability of intracellular iron. To learn more about iron metabolism in L. pneumophila, we have undertaken an analysis of the iron proteins of the bacterium. We first developed an assay to identify proteins by 59Fe labelling and nondenaturing polyacrylamide gel electrophoresis. The assay revealed seven iron proteins (IPs) with apparent molecular weights of 500, 450, 250, 210, 150, 130, and 85. IP150 comigrates with superoxide dismutase activity and is probably the Fe-superoxide dismutase of L. pneumophila. IP210 is the major iron-containing protein (MICP). To identify and characterize MICP, we purified the protein and cloned and sequenced its gene. MICP is a monomeric protein containing 891 amino acids, and it has a calculated molecular mass of 98,147 Da. Analysis of the sequence revealed that MICP has two interesting homologies. First, MICP is highly homologous with the human iron-responsive element-binding protein, consistent with the hypothesis that this critical iron-regulatory molecule of humans has a prokaryotic ancestor. Second, MICP is highly homologous with the Escherichia coli aconitase and to a lesser extent with porcine heart mitochondrial aconitase. Consistent with this, we found that MICP exhibits aconitase activity. In contrast to other aconitases, MICP has a single amino acid change of a potentially deleterious type at a site thought to be critical for substrate binding and enzymatic activity. However, the specific activity of MICP is roughly comparable to that of other aconitases, suggesting that the mutation has at most a mild effect on the aconitase activity of MICP. The abundance of MICP in L. pneumophila suggests either that L. pneumophila requires high aconitase and perhaps tricarboxylic acid cycle activity or that the bacterium requires large amounts of this protein to serve an additional role in bacterial physiology. A need for large amounts of MICP, which contains four Fe atoms per molecule when fully loaded, could at least partly explain L. pneumophila's high metabolic requirement for iron.     

A developmentally regulated aconitase related to iron-regulatory protein-1 is localized in the cytoplasm and in the mitochondrion of Trypanosoma brucei

Mitochondrial energy metabolism and Krebs cycle activities are developmentally regulated in the life cycle of the protozoan parasite Trypanosoma brucei. Here we report cloning of a T. brucei aconitase gene that is closely related to mammalian iron-regulatory protein 1 (IRP-1) and plant aconitases. Kinetic analysis of purified recombinant TbACO expressed in Escherichia coli resulted in a K(m) (isocitrate) of 3 +/- 0.4 mM, similar to aconitases of other organisms. This was unexpected since an arginine conserved in the aconitase protein family and crucial for substrate positioning in the catalytic center and for activity of pig mitochondrial aconitase (Zheng, L., Kennedy, M. C., Beinert, H., and Zalkin, H. (1992) J. Biol. Chem. 267, 7895-7903) is substituted by leucine in the TbACO sequence. Expression of the 98-kDa TbACO was shown to be lowest in the slender bloodstream stage of the parasite, 8-fold elevated in the stumpy stage, and increased a further 4-fold in the procyclic stage. The differential expression of TbACO protein contrasted with only minor changes in TbACO mRNA, indicating translational or post-translational mechanisms of regulation. Whereas animal cells express two distinct compartmentalized aconitases, mitochondrial aconitase and cytoplasmic aconitase/IRP-1, TbACO accounts for total aconitase activity in trypanosomes. By cell fractionation and immunofluorescence microscopy, we show that native as well as a transfected epitope-tagged TbACO localizes in both the mitochondrion (30%) and in the cytoplasm (70%). Together with phylogenetic reconstructions of the aconitase family, this suggests that animal IRPs have evolved from a multicompartmentalized ancestral aconitase. The possible functions of a cytoplasmic aconitase in trypanosomes are discussed.     

The AraC-type regulator RipA represses aconitase and other iron proteins from Corynebacterium under iron limitation and is itself repressed by DtxR

The mRNA level of the aconitase gene acn of Corynebacterium glutamicum is reduced under iron limitation. Here we show that an AraC-type regulator, termed RipA for "regulator of iron proteins A," is involved in this type of regulation. A C. glutamicum DeltaripA mutant has a 2-fold higher aconitase activity than the wild type under iron limitation, but not under iron excess. Comparison of the mRNA profiles of the DeltaripA mutant and the wild type revealed that the acn mRNA level was increased in the DeltaripA mutant under iron limitation, but not under iron excess, indicating a repressor function of RipA. Besides acn, some other genes showed increased mRNA levels in the DeltaripA mutant under iron starvation (i.e. those encoding succinate dehydrogenase (sdhCAB), nitrate/nitrite transporter and nitrate reductase (narKGHJI), isopropylmalate dehydratase (leuCD), catechol 1,2-dioxygenase (catA), and phosphotransacetylase (pta)). Most of these proteins contain iron. Purified RipA binds to the upstream regions of all operons mentioned above and in addition to that of the catalase gene (katA). From 13 identified binding sites, the RipA consensus binding motif RRGCGN(4)RYGAC was deduced. Expression of ripA itself is repressed under iron excess by DtxR, since purified DtxR binds to a well conserved binding site upstream of ripA. Thus, repression of acn and the other target genes indicated above under iron limitation involves a regulatory cascade of two repressors, DtxR and its target RipA. The modulation of the intracellular iron usage by RipA supplements mechanisms for iron acquisition that are directly regulated by DtxR.     

Cloning of the aconitase gene (acn) of Escherichia coli K12

Lambda phages containing the aconitase gene (acn) of Escherichia coli K12 have been isolated by hybridization with an M13 probe containing part of the aconitase gene (citB) of Bacillus subtilis. Aconitase specific activities are amplified 5- to 18-fold in thermally induced lambda acn lysogens and threefold in a strain transformed with a plasmid derivative (pGS181).     

Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12

The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA).     

Deduced amino acid sequence of Escherichia coli adenosine deaminase reveals evolutionarily conserved amino acid residues: implications for catalytic function

The goal of the research reported here is to identify evolutionarily conserved amino acid residues associated with enzymatic deamination of adenosine. To do this, we isolated molecular clones of the Escherichia coli adenosine deaminase gene by functional complementation of adenosine deaminase deficient bacteria and deduced the amino acid sequence of the enzyme from the nucleotide sequence of the gene. Nucleotide sequence analysis revealed the presence of a 996-nucleotide open reading frame encoding a protein of 332 amino acids having a molecular weight of 36,345. The deduced amino acid sequence of the E. coli enzyme has approximately 33% identity with those of the mammalian adenosine deaminases. With conservative amino acid substitutions the overall sequence homology approaches 50%, suggesting that the structures and functions of the mammalian and bacterial enzymes are similar. Additional amino acid sequence analysis revealed specific residues that are conserved among all three adenosine deaminases and four AMP deaminases for which sequence information is currently available. In view of previously published enzymological data and the conserved amino acid residues identified in this study, we propose a model to account for the enzyme-catalyzed hydrolytic deamination of adenosine. Potential catalytic roles are assigned to the conserved His 214, Cys 262, Asp 295, and Asp 296 residues of mammalian adenosine deaminases and the corresponding conserved amino acid residues in bacterial adenosine deaminase and the eukaryotic AMP deaminases.     

The phosphinomethylmalate isomerase gene pmi, encoding an aconitase-like enzyme, is involved in the synthesis of phosphinothricin tripeptide in Streptomyces viridochromogenes

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197-222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.     

Binding of cytosolic aconitase to the iron responsive element of porcine mitochondrial aconitase mRNA.

The 5' end of porcine mitochondrial aconitase mRNA contains an iron responsive element (IRE)-like secondary structure (T. Dandekar, R. Stripecke, N. K. Gray, B. Goosen, A. Constable, H. E. Johansson, and M. W. Hentze (1991) EMBO J. 10, 1903-1909). A protein from a liver extract binds to a mitochondrial aconitase RNA probe and supports the identification of this sequence as an IRE. Purified cytosolic aconitase but not the mitochondrial enzyme binds to this IRE as well as to a ferritin IRE. All forms of cytosolic aconitase, [4Fe-4S] enzyme, [3Fe-4S] enzyme and apoenzyme bind with similar affinity. A Kd of 0.25 nM was calculated for the apoaconitase-IRE interaction from Scatchard analysis. These results support the conclusion that cytosolic aconitase is an IRE-binding protein which may regulate translation of mitochondrial aconitase mRNA.     

Cloning and sequencing of the gltX gene, encoding the glutamyl-tRNA synthetase of Rhizobium meliloti A2.

The gltX gene, coding for the glutamyl-tRNA synthetase of Rhizobium meliloti A2, was cloned by using as probe a synthetic oligonucleotide corresponding to the amino acid sequence of a segment of the glutamyl-tRNA synthetase. The codons chosen for this 42-mer were those most frequently used in a set of R. meliloti genes. DNA sequence analysis revealed an open reading frame of 484 codons, encoding a polypeptide of Mr 54,166 containing the amino acid sequences of an NH2-terminal and various internal fragments of the enzyme. Compared with the amino acid sequence of the glutamyl-tRNA synthetase of Escherichia coli, the N-terminal third of the R. meliloti enzyme was strongly conserved (52% identity); the second third was moderately conserved (38% identity) and included a few highly conserved segments, whereas no significant similarity was found in the C-terminal third. These results suggest that the C-terminal part of the protein is probably not involved in the recognition of substrates, a feature shared with other aminoacyl-tRNA synthetases.     

Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8.

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.     

Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8.

The gene coding for isocitrate dehydrogenase of an extreme thermophile, Thermus thermophilus HB8, was cloned and sequenced. This gene consists of a single open reading frame of 1,485 bp preceded by a Shine-Dalgarno ribosome binding site. Promoter- and terminatorlike sequences were detected upstream and downstream of the open reading frame, respectively. The G + C content of the coding region was 65.6%, and that of the third nucleotide of the codons was 90.3%. On the basis of the deduced amino acid sequence, the Mr of the monomeric enzyme was calculated as 54,189, an Mr which is similar to that of the purified protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison of the amino acid sequence of the T. thermophilus enzyme with that of the Escherichia coli enzyme showed (i) a 37% overall similarity; (ii) the conservation of the Ser residue, which is known to be phosphorylated in the E. coli enzyme, and of the surrounding sequence; and (iii) the presence of 141 extra residues at the C terminus of the T. thermophilus enzyme. T. thermophilus isocitrate dehydrogenase showed a high sequence homology (33% of the amino acid sequence is identical) to isopropylmalate dehydrogenase from the same organism and was suggested to have evolved from a common ancestral enzyme.