Aconitase/Iron-responsive element-binding protein 2 (Aconitase/IRP2)
Type: Family

This entry represents Aconitate hydratase A found predominantly in bacteria. Iron-responsive element-binding protein 2 and Cytoplasmic aconitate hydratase from animals; and Aconitate hydratase from plants also belong to this family. It also includes Phosphinomethylmalate isomerase (Pmi) involved in the biosynthesis of phosphinothricin tripeptide, a natural-product antibiotic and potent herbicide . Pmi has no aconitase activity with citrate as a substrate and is not able to complement an acnA mutant .

Aconitase (aconitate hydratase;

) is an iron-sulphur protein that contains a [4Fe-4S]-cluster and catalyses the interconversion of isocitrate and citrate via a cis-aconitate intermediate. Aconitase functions in both the TCA and glyoxylate cycles, however unlike the majority of iron-sulphur proteins that function as electron carriers, the [4Fe-4S]-cluster of aconitase reacts directly with an enzyme substrate. In eukaryotes there is a cytosolic form (cAcn) and a mitochondrial form (mAcn) of the enzyme. In bacteria there are also 2 forms, aconitase A (AcnA) and B (AcnB). Several aconitases are known to be multi-functional enzymes with a second non-catalytic, but essential function that arises when the cellular environment changes, such as when iron levels drop . Eukaryotic cAcn and mAcn, and bacterial AcnA have the same domain organisation, consisting of three N-terminal α/β/α domains, a linker region, followed by a C-terminal 'swivel' domain with a β/β/α structure (1-2-3-linker-4), although mAcn is smaller than cAcn. However, bacterial AcnB has a different organisation: it contains an N-terminal HEAT-like domain, followed by the 'swivel' domain, then the three α/β/α domains (HEAT-4-1-2-3) .

Eukaryotic cAcn enzyme balances the amount of citrate and isocitrate in the cytoplasm, which in turn creates a balance between the amount of NADPH generated from isocitrate by isocitrate dehydrogenase with the amount of acetyl-CoA generated from citrate by citrate lyase. Fatty acid synthesis requires both NADPH and acetyl-CoA, as do other metabolic processes, including the need for NADPH to combat oxidative stress. The enzymatic form of cAcn predominates when iron levels are normal, but if they drop sufficiently to cause the disassembly of the [4Fe-4S]-cluster, then cAcn undergoes a conformational change from a compact enzyme to a more open L-shaped protein known as iron regulatory protein 1 (IRP1; or IRE-binding protein 1, IREBP1) . As IRP1, the catalytic site and the [4Fe-4S]-cluster are lost, and two new RNA-binding sites appear. IRP1 functions in the post-transcriptional regulation of genes involved in iron metabolism -it binds to mRNA iron-responsive elements (IRE), 30-nucleotide stem-loop structures at the 3' or 5' end of specific transcripts. Transcripts containing an IRE include ferritin L and H subunits (iron storage), transferrin (iron plasma chaperone), transferrin receptor (iron uptake into cells), ferroportin (iron exporter), mAcn, succinate dehydrogenase, erythroid aminolevulinic acid synthetase (tetrapyrrole biosynthesis), among others. If the IRE is in the 5'-UTR of the transcript (e.g. in ferritin mRNA), then IRP1-binding prevents its translation by blocking the transcript from binding to the ribosome. If the IRE is in the 3'-UTR of the transcript (e.g. transferrin receptor), then IRP1-binding protects it from endonuclease degradation, thereby prolonging the half-life of the transcript and enabling it to be translated .

IRP2 is another IRE-binding protein that binds to the same transcripts as IRP1. However, since IRP1 is predominantly in the enzymatic cAcn form, it is IRP2 that acts as the major metabolic regulator that maintains iron homeostasis . Although IRP2 is homologous to IRP1, IRP2 lacks aconitase activity, and is known only to have a single function in the post-transcriptional regulation of iron metabolism genes . In iron-replete cells, IRP2 activity is regulated primarily by iron-dependent degradation through the ubiquitin-proteasomal system.

Bacterial AcnB is also known to be multi-functional. In addition to its role in the TCA cycle, AcnB was shown to be a post-transcriptional regulator of gene expression in Escherichia coli and Salmonella enterica . In S. enterica, AcnB initiates a regulatory cascade controlling flagella biosynthesis through an interaction with the ftsH transcript, an alternative RNA polymerase sigma factor. This binding lowers the intracellular concentration of FtsH protease, which in turn enhances the amount of RNA polymerase sigma32 factor (normally degraded by FtsH protease), and sigma32 then increases the synthesis of chaperone DnaK, which in turn promotes the synthesis of the flagellar protein FliC. AcnB regulates the synthesis of other proteins as well, such as superoxide dismutase (SodA) and other enzymes involved in oxidative stress.