Aconitase

Aconitase, a moonlighting enzyme

Aconitase (EC 4.2.1.3) is an iron-sulphur protein that catalyses the interconversion of citrate and isocitrate, via a cis-aconitate intermediate, functioning in both the tricarboxylic acid (TCA) and glyoxylate cycles. In eukaryotes, there is a cytosolic form (cAcn) and a mitochondrial form (mAcn), which are encoded by separate genes. In bacteria there are also two forms, aconitase A (AcnA) that resembles mAcn, and aconitase B (AcnB). Different moonlighting activities have been shown for cAcn, mAcn and AcnB.

Cytosolic aconitase

cAcn interconverts citrate and isocitrate in the cytoplasm, allowing the cell to balance 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 these products, NADPH and acetyl-CoA, as do other metabolic processes. In addition, the enzymatic activity of cAcn appears to contribute to cellular defence against oxidative stress by providing a source of isocitrate for NADPH production; glutathione reductase uses NADPH to regenerate glutathione after it has been oxidised by glutathione peroxidase as a means of removing oxidative stress-induced peroxide (H2O2).

However, aconitase only has catalytic activity when iron concentrations are high (at normal levels). If the iron concentrations drop too low, then cAcn loses it [4Fe-4S]-cluster and its enzymatic activity, and takes on a new moonlighting role. It is even given a new name, iron regulatory protein 1 or IRP1 (also called iron response element-binding protein 1, or IREP1). When cAcn switches to become IRP1, it gains the ability to bind mRNA transcripts involved in the uptake, storage and utilisation of iron. Once bound to these transcripts, IRP1 exerts post-transcription regulation by either promoting or preventing their translation.

Another iron regulatory protein, IRP2, lacks aconitase activity but is the major iron metabolic regulator that maintains iron homeostasis, binding to the same mRNAs as IRP1. By contrast, IRP1 acts predominantly as an aconitase, switching to IRP1 under high oxygen levels, possibly under conditions of infection or inflammation, where the production of reactive oxygen species and nitric oxide could destabilise the labile iron-sulphur cluster.

Mitochondrial aconitase

mAcn plays a key role in carbohydrate metabolism, catalysing the second step of the TCA cycle to produce isocitrate from citrate. In addition to energy production from the breakdown of carbohydrates, several intermediates of the TCA cycle are also biosynthetic precursors of haem and amino acids.

mAcn also moonlights: it has a second essential role - in mitochondrial DNA (mtDNA) maintenance. mAcn acts to stabilise mtDNA, forming part of mtDNA protein-DNA complexes known as nucleoids. It is thought to reversibly model nucleoids to directly influence mitochondrial gene expression in response to changes in the cellular environment.

These two functions are linked. The function of the TCA cycle is to provide high energy electrons in the form of NADH and FADH2 to the mitochondrial oxidative phosphorylation pathway, while mtDNA encodes components of the oxidative phosphorylation pathway. As such, mAcn could act as a metabolic regulator to couple energy metabolism with mitochondrial gene expression.

Bacterial aconitase B

Like mAcn, AcnB is a major TCA cycle enzyme with a second important role to play. In Escherichia coli, AcnB was shown to be a post-transcriptional regulator of gene expression. In Salmonella 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.

AcnB has a different domain architecture from cAcn or mAcn, and differs from other aconitases by containing a HEAT-like domain. HEAT-like domains are thought to be involved in protein-protein interactions. The N-terminal region, which contains both the HEAT-like domain and a ‘swivel’ domain, was shown to be sufficient for dimerisation and for AcnB binding to mRNA. An iron-mediated dimerisation mechanism may be responsible for switching AcnB between its catalytic and regulatory roles, as dimerisation requires iron while mRNA binding is inhibited by iron.

Escherichia coli also possess aconitase A (AcnA), which is a stress-induced enzyme synthesised during the stationary phase of growth. AcnA is similar in domain architecture to eukaryotic mAcn.

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