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.     

Inactivation of the tricarboxylic acid cycle aconitase gene from Streptomyces viridochromogenes Tü494 impairs morphological and physiological differentiation

The tricarboxylic acid (TCA) cycle aconitase gene acnA from Streptomyces viridochromogenes Tü494 was cloned and analyzed. AcnA catalyzes the isomerization of citrate to isocitrate in the TCA cycle, as indicated by the ability of acnA to complement the aconitase-deficient Escherichia coli mutant JRG3259. An acnA mutant was unable to develop aerial mycelium and to sporulate, resulting in a bald phenotype. Furthermore, the mutant did not produce the antibiotic phosphinothricin tripeptide, demonstrating that AcnA also affects physiological differentiation.     

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.     

Phosphinothricin-tripeptide biosynthesis: An original version of bacterial secondary metabolism?

Streptomyces viridochromogenes Tü494 produces the herbicide phosphinothricyl-alanyl-alanine (phosphinothricin-tripeptide = PTT; bialaphos). Its bioactive moiety phosphinothricin competitively inhibits bacterial and plant glutamine synthetases. The biosynthesis of PTT includes the synthesis of the unusual amino acid N-acetyl-demethyl-phosphinothricin and a three step condensation via non-ribosomal peptide synthetases. Two characteristics within the PTT biosynthesis make it suitable to study the evolution of secondary metabolism biosynthesis. First, PTT biosynthesis represents the only known system where all peptide synthetase modules are located on separate proteins. This ‘single enzyme system’ might be an archetype of the multimodular and multienzymatic non-ribosomal peptide synthetases in evolutionary terms. The second interesting feature of PTT biosynthesis is the pathway-specific aconitase Pmi that is involved in the supply of N-acetyl-demethyl-phosphinothricin. Pmi is highly similar to the tricarboxylic acid aconitase AcnA. They share 64% identity at the DNA level and both belong to the Iron-Regulatory-Protein/AcnA family. Despite their high sequence similarity, AcnA and Pmi catalyze different reactions and are not able to substitute for each other. Thus, the enzyme pair AcnA/Pmi presents an example of the evolution of a secondary metabolite-specific enzyme from a primary metabolism enzyme.     

Contrasting sensitivities of Escherichia coli aconitases A and B to oxidation and iron depletion

Superoxide damages dehydratases that contain catalytic [4Fe-4S](2+) clusters. Aconitases are members of that enzyme family, and previous work showed that most aconitase activity is lost when Escherichia coli is exposed to superoxide stress. More recently it was determined that E. coli synthesizes at least two isozymes of aconitase, AcnA and AcnB. Synthesis of AcnA, the less-abundant enzyme, is positively controlled by SoxS, a protein that is activated in the presence of superoxide-generating chemicals. We have determined that this arrangement exists because AcnA is resistant to superoxide in vivo. Surprisingly, purified AcnA is extremely sensitive to superoxide and other chemical oxidants unless it is combined with an uncharacterized factor that is present in cell extracts. In contrast, AcnB is highly sensitive to a variety of chemical oxidants in vivo, in extracts, and in its purified form. Thus, the induction of AcnA during oxidative stress provides a mechanism to circumvent a block in the tricarboxylic acid cycle. AcnA appears to be as catalytically competent as AcnB, so the retention of the latter as the primary housekeeping enzyme must provide some other advantage. We observed that the [4Fe-4S] cluster of AcnB is in dynamic equilibrium with the surrounding iron pool, so that AcnB is rapidly demetallated when intracellular iron pools drop. AcnA and other dehydratases do not show this trait. Demetallated AcnB is known to bind its cognate mRNA. The absence of AcnB activity also causes the accumulation and excretion of citrate, an iron chelator for which E. coli synthesizes a transport system. Thus, AcnB may be retained as the primary aconitase because the lability of its exposed cluster allows E. coli to sense and respond to iron depletion.     

A Novel A3 Group Aconitase Tolerates Oxidation and Nitric Oxide

Achromobacter denitrificans YD35 is an NO₂⁻-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 mm NO₂⁻. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10⁻³ min⁻¹, which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S]²⁺ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO₂⁻ levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10⁻³ min⁻¹, which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO₂⁻, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO₂⁻ tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO₂⁻ milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO₂⁻ tolerance mechanism in this strain.     

Involvement of the Iron Regulatory Protein from Eisenia andrei Earthworms in the Regulation of Cellular Iron Homeostasis

Iron homeostasis in cells is regulated by iron regulatory proteins (IRPs) that exist in different organisms. IRPs are cytosolic proteins that bind to iron-responsive elements (IREs) of the 5′- or 3′-untranslated regions (UTR) of mRNAs that encode many proteins involved in iron metabolism. In this study, we have cloned and described a new regulatory protein belonging to the family of IRPs from the earthworm Eisenia andrei (EaIRP). The earthworm IRE site in 5′-UTR of ferritin mRNA most likely folds into a secondary structure that differs from the conventional IRE structures of ferritin due to the absence of a typically unpaired cytosine that participates in protein binding. Prepared recombinant EaIRP and proteins from mammalian liver extracts are able to bind both mammalian and Eisenia IRE structures of ferritin mRNA, although the affinity of the rEaIRP/Eisenia IRE structure is rather low. This result suggests the possible contribution of a conventional IRE structure. When IRP is supplemented with a Fe-S cluster, it can function as a cytosolic aconitase. Cellular cytosolic and mitochondrial fractions, as well as recombinant EaIRP, exhibit aconitase activity that can be abolished by the action of oxygen radicals. The highest expression of EaIRP was detected in parts of the digestive tract. We can assume that earthworms may possess an IRE/IRP regulatory network as a potential mechanism for maintaining cellular iron homeostasis, although the aconitase function of EaIRP is most likely more relevant.     

Molecular characterization of the iron binding protein ferritin in Eisenia andrei earthworms

Ferritin is a storage protein that plays a key role in iron metabolism. In this study, we report on the sequence characterization of a ferritin-coding cDNA in Eisenia andrei earthworms isolated by RT-PCR using degenerated primers, and we suggest the presence of a putative IRE in the 5′-UTR of ferritin mRNA. The obtained ferritin sequence was compared with those of other animals showing sequence and structure homology in consensus sites, including the iron-responsive element (IRE) and ferroxidase centers. Despite the sequence homology in the E. andrei mRNA of ferritin with the sequences of other animals in consensus IRE sites, the presented cytosine in the IRE of E. andrei ferritin in the expected position does not form a conventional bulge. The presence of ferritin in the coelomic fluid of E. andrei was proven by iron staining assay. Moreover, aconitase activity in the coelomic fluid was assessed by aconitase assay, suggesting the presence of an iron regulatory protein. Quantitative analysis revealed changes in the gene expression levels of ferritin in coelomocytes in response to bacterial challenge, reaching the maximum level 8 h after the stimulation with both Gram-positive and Gram-negative bacteria.     

Nucleotide-Specific Recognition of Iron-Responsive Elements by Iron Regulatory Protein 1

IRP1 [iron regulatory protein (IRP) 1] is a bifunctional protein with mutually exclusive end-states. In one mode of operation, IRP1 binds iron-responsive element (IRE) stem–loops in messenger RNAs encoding proteins of iron metabolism to control their rate of translation. In its other mode, IRP1 serves as cytoplasmic aconitase to correlate iron availability with the energy and oxidative stress status of the cell. IRP1/IRE binding occurs through two separate interfaces, which together contribute about two-dozen hydrogen bonds. Five amino acids make base-specific contacts and are expected to contribute significantly to binding affinity and specificity of this protein:RNA interaction. In this mutagenesis study, each of the five base-specific amino acids was changed to alter binding at each site. Analysis of IRE binding affinity and translational repression activity of the resulting IRP1 mutants showed that four of the five contact points contribute uniquely to the overall binding affinity of the IRP1:IRE interaction, while one site was found to be unimportant. The stronger-than-expected effect on binding affinity of mutations at Lys379 and Ser681, residues that make contact with the conserved nucleotides G16 and C8, respectively, identified them as particularly critical for providing specificity and stability to IRP1:IRE complex formation. We also show that even though the base-specific RNA-binding residues are not part of the aconitase active site, their substitutions can affect the aconitase activity of holo-IRP1, positively or negatively.     

Inactivation of both RNA binding and aconitase activities of iron regulatory protein-1 by quinone-induced oxidative stress

Iron regulatory protein-1 (IRP-1) controls the expression of several mRNAs by binding to iron-responsive elements (IREs) in their untranslated regions. In iron-replete cells, a 4Fe-4S cluster converts IRP-1 to cytoplasmic aconitase. IRE binding activity is restored by cluster loss in response to iron starvation, NO, or extracellular H2O2. Here, we study the effects of intracellular quinone-induced oxidative stress on IRP-1. Treatment of murine B6 fibroblasts with menadione sodium bisulfite (MSB), a redox cycling drug, causes a modest activation of IRP-1 to bind to IREs within 15-30 min. However, IRE binding drops to basal levels within 60 min. Surprisingly, a remarkable loss of both IRE binding and aconitase activities of IRP-1 follows treatment with MSB for 1-2 h. These effects do not result from alterations in IRP-1 half-life, can be antagonized by the antioxidant N-acetylcysteine, and regulate IRE-containing mRNAs; the capacity of iron-starved MSB-treated cells to increase transferrin receptor mRNA levels is inhibited, and MSB increases the translation of a human growth hormone indicator mRNA bearing an IRE in its 5'-untranslated region. Nonetheless, MSB inhibits ferritin synthesis. Thus, menadione-induced oxidative stress leads to post-translational inactivation of both genetic and enzymatic functions of IRP-1 by a mechanism that lies beyond the "classical" Fe-S cluster switch and exerts multiple effects on cellular iron metabolism.     

Expression of phosphinothricin acetyltransferase from the root specific par promoter in transgenic tobacco plants is sufficient for herbicide tolerance

Summary The pat gene, coding for phosphinothricin acetyltransferase (PAT) from Streptomyces viridochromogenes, was cloned behind the par promoter of the hemoglobin gene from Parasponia andersonii, Introduction into tobacco (Nicotiana tabacum) resulted in predominantly root specific PAT expression. Application of 5 l/ha BASTA^® (herbicidal component: phosphinothricin) did not effect growth morphology and vigor of the plants. After application of 20 l/ha BASTA^® the plants showed herbicide damage. Nevertheless, they all recovered by forming new undamaged leaves and resumed full growth despite virtually non-detectable expression of the PAT enzyme in the leaves.Abbreviations BAP 6-benzylaminopurine - CaMV Cauliflower Mosaic Virus - IAA indole-3-acetic acid - kb kilobases - LB Luria-Bertani - MS Murashige and Skoog - par Parasponia andersonii - PAT phosphinothricin acetyltransferase - ppt phosphinothricin - TCA trichloric acid     

Cooperativity of two active sites in bacterial homodimeric aconitases

Aconitase catalyzes a reversible isomerization of citrate into isocitrate in the Krebs cycle. Escherichia coli possesses two kinds of aconitases, aconitase A (AcnA) and B (AcnB), whose structural organizations are different. We analyzed the structural state of AcnA by the chemical crosslinking and small-angle X-ray scattering. The protein adopts a homodimer in solution, as AcnB does. The catalytic assay of the two aconitases revealed that the isomerization of isocitrate displayed a negative cooperativity of the two active sites within each homodimer. On the other hand, insignificant cooperativity was observed in the reverse reaction. Therefore, the homodimerization of AcnAB yields a substrate-dependent cooperative effect. In conjunction with the dissociable homodimer of AcnB, the catalytic property could affect the intracellular metabolic process involving the Krebs cycle.     

The genes that encode the gonococcal transferrin binding proteins,TbpB and TbpA,are differentially regulated by MisR under iron‐replete and iron‐depleted conditions

Neisseria gonorrhoeae produces two transferrin binding proteins, TbpA and TbpB, which together enable efficient iron transport from human transferrin. We demonstrate that expression of the tbp genes is controlled by MisR, a response regulator in the two‐component regulatory system that also includes the sensor kinase MisS. The tbp genes were up‐regulated in the misR mutant under iron‐replete conditions but were conversely down‐regulated in the misR mutant under iron‐depleted conditions. The misR mutant was capable of transferrin‐iron uptake at only 50% of wild‐type levels, consistent with decreased tbp expression. We demonstrate that phosphorylated MisR specifically binds to the tbpBA promoter and that MisR interacts with five regions upstream of the tbpB start codon. These analyses confirm that MisR directly regulates tbpBA expression. The MisR binding sites in the gonococcus are only partially conserved in Neisseria meningitidis, which may explain why tbpBA was not MisR‐regulated in previous studies using this related pathogen. This is the first report of a trans‐acting protein factor other than Fur that can directly contribute to gonococcal tbpBA regulation.