The shikimate biosynthetic pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites; it involves seven enzymatic steps in which phosphoenolpyruvate and erythrose 4-phosphate are converted into chorismate. In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin. As mammals do not possess such a biosynthetic system, the enzymes involved in the pathway have aroused considerable interest as potential targets for the development of antimicrobial drugs and herbicides. As an initiative to investigate the mechanism of some of these enzymes, we showed that the antimicrobial effect of (6S)-6-fluoroshikimate is the result of irreversible inhibition of 4-amino-4-deoxychorismate synthase by 2-fluorochorismate. Based on this study, a catalytic mechanism for this enzyme was proposed, in which the residue Lys-274 is involved in the formation of a covalent intermediate. In another study, Yersinia enterocolitica Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin, was for the first time biochemically characterized and shown to catalyse the formation of salicylate from chorismate via isochorismate as a reaction intermediate. A three-dimensional model for this enzyme was constructed that will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake.
The shikimate biosynthetic pathway involves seven enzymatic steps in which phosphoenolpyruvate and erythrose-4-phosphate are converted into chorismate . In Escherichia coli, five chorismate-utilizing enzymes catalyse the synthesis of aromatic compounds such as L-phenylalanine, L-tyrosine, L-tryptophan, folate, ubiquinone and siderophores such as yersiniabactin and enterobactin . The shikimate pathway is utilized in algae, higher plants, bacteria, fungi and apicomplexan parasites, but it is absent in mammals. The enzymes involved in the pathway therefore provide attractive targets for the development of antimicrobial drugs and effective herbicides [3,4]. As part of an effort to understand the molecular basis for the interesting similarities and striking differences seen in chorismate-utilizing enzymes, this work describes mechanistic and inhibition studies carried out particularly on ADC (4-amino-4-deoxychorismate) synthase, which catalyses the reaction of chorismate and ammonia to ADC.
Several possible mechanisms have previously been proposed for the reactions catalysed by ADC synthase, anthranilate synthase [which catalyses the formation of o-aminobenzoate (anthranilate) from chorismate] and isochorismate synthase, which catalyses the interconversion of chorismate and isochorismate  (Scheme 1). However, until recently there had been limited detailed mechanistic information available on any of the three enzymes, which show some protein sequence homology (approx. 20%) but are a functionally distinct family of enzymes. The crystal structures of anthranilate synthase from three different organisms [6–8], and the PabB subunit of E. coli ADC synthase , showed that these two enzymes have very similar active sites, but also revealed some variations in the active site structures that may lead to the different reactions that these enzymes catalyse. Our research focused on elucidating the conserved mechanistic features of the enzymes, and exploring the basis for diversity in the reactions catalysed.
Here, we also report the biochemical characterization of Yersinia enterocolitica Irp9, which catalyses the conversion of chorismate into salicylate, a building block in the biosynthesis of the siderophore yersiniabactin. Mechanistic studies of this bacterial salicylate synthase identified the intermediate isochorismate as being involved in the formation of salicylate. The mechanistic significance of this finding provides important clues to understanding key aspects common to the mechanisms of mono- and bi-functional chorismate-utilizing enzymes.
A unifying mechanism for chorismate-utilizing enzymes using ADC synthase as a case study
A key feature of the active site of ADC synthase is a lysine residue at a position which is conserved as an alanine in anthranilate synthase and isochorismate synthase. The ϵ-amino group of the lysine is predicted to sit in close proximity to C-2 of chorismate. Toney and co-workers  explored the mechanistic role of this lysine (Lys-274) in a site-directed mutagenesis study of E. coli ADC synthase. A K274A mutant was found to produce a mixture of the expected product ADC and the intermediate of the anthranilate synthase reaction, 2-amino-2-deoxyisochorismate. The result of that study provides strong evidence that the first step of the mechanism of all three enzymes is conserved, and is an attack of a nucleophile on C-2 of chorismate. This leads to elimination of the C4 hydroxy group via an SN2″ mechanism. The nature of the nucleophile varies between the enzymes, with an active site residue (Lys-274), ammonia or water attacking in the reactions catalysed by ADC synthase, anthranilate synthase and isochorismate synthase respectively. After this initial conserved step, the reactions catalysed by the enzymes diverge.
For ADC synthase, a key feature of the proposed mechanism is the formation of a covalent enzyme-bound intermediate, which is turned over by the secondary attack of ammonia at C4 (Figure 1a). Evidence for the formation of this covalent intermediate was obtained in our laboratory during studies of the mechanism of action of the antibacterial agent (6S)-6-fluoroshikimate [11,12]. This compound was shown to be converted into 2-fluorochorismate by enzymes of the shikimate pathway [13,14]. 2-Fluorochorismate was subsequently found to inactivate ADC synthase via an irreversible covalent modification of Lys-274 . The formation of this irreversible covalent modification is thought to mimic the native mechanism of the enzyme (Figure 1b).
More recently, we have used electrospray mass spectrometry to directly detect the formation of the covalent intermediate on the PabB subunit of ADC synthase during catalysis . On treatment of ADC synthase with chorismate, the formation of a 208 Da covalent modification was observed on the PabB subunit. The mass of this modification is in accordance with that expected for the enzyme-bound covalent intermediate proposed by Toney and co-workers .
Salicylate synthase: a bifunctional enzyme involved in the biosynthesis of the siderophore yersiniabactin
Almost all cells and organisms require iron as a cofactor or prosthetic group for essential enzymes that are involved in many basic cellular functions and metabolic pathways . Iron is a major component of the Earth's crust, but its benefits are balanced by severe limitations to its biological utility. This is because under aerobic, aqueous and neutral pH conditions, iron is present essentially in the oxidized ferric form (Fe3+), which aggregates into insoluble oxy-hydroxide polymers . Moreover, when reduced, Fe2+ catalyses the formation of oxygen free radicals that are highly toxic. To deal with these difficulties, host organisms have evolved solubilizing iron-transport and -storage systems, such as carrier proteins (transferrin, lactoferrin and ferritin) or the protoporphyrin ring in haemoproteins. As a result, iron homoeostasis is so strictly regulated that there is virtually no free iron in living organisms . For example, the concentration of free Fe3+ in human serum is extremely low (10−9–10−15 M).
Iron is essential for growth of pathogenic bacteria, and during infection, bacteria must obtain iron from host stores to support their normal metabolism . A common strategy used by many pathogenic micro-organisms to acquire iron as well as tackling the problem of low iron bioavailability is to produce siderophores, which are low-molecular-mass organic chelators designed to sequester ferric iron with high affinity. Excreted into the environment, the siderophores bind ferric ions and deliver them back to the cytoplasm of the micro-organism via specific membrane receptors and transport proteins .
For many pathogenic bacteria, the efficiency of iron uptake is directly related to virulence; therefore the inhibition of bacterial iron uptake has long been considered a promising area of research towards the creation of novel therapeutic options. Virulence in Yersinia pestis, the causative agent of bubonic plague, has been correlated with the biosynthesis and transport of the siderophore yersiniabactin, which chelates ferric iron with a dissociation constant of 10−36 M through the nitrogen atoms of its thazoline rings . The Y. pestis yersiniabactin genes share almost 100% identity with those of Y. enterocolitica, which, unlike the vector-borne pathogen Y. pestis, is an enteric pathogen that infects humans through ingestion of contaminated food or water, and can cause infections such as gastroenteritis. The locus involved in assembly of yersiniabactin in Y. enterocolitica has been genetically characterized, and includes nine irp genes [23–27].
Formation of yersiniabactin occurs via a hybrid non-ribosomal peptide synthetase/polyketide synthase system  that assembles the siderophore from the precursor salicylic acid, a linker group derived from malonyl-CoA, three molecules of cysteine and three methyl groups from S-adenosylmethionine . In bacteria, it was shown that salicylate can be derived from chorismate via isochorismate (Figure 2). For example, in Pseudomonas aeruginosa and Ps. fluorescens, two genes coding for an isochorismate synthase and an isochorismate pyruvate lyase have been identified as being responsible for the biosynthesis of salicylate.
It has been postulated, using genetic complementation studies, that Irp9, which is involved in the biosynthesis of the siderophore yersiniabactin in Y. enterocolitica, converts chorismate directly into salicylate . We have now characterized Irp9 biochemically by cloning the corresponding gene and heterologously expressing the corresponding protein in E. coli. 1H NMR analysis of the Irp9-catalysed reaction demonstrated that Irp9 does form salicylate from chorismate via the intermediate isochorismate .
Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase . It is worth noting that the same kinds of inhibitors were also tested against a bifunctional PabA/PabB from Arabidopsis thaliana , with the most potent inhibitors having inhibition constants of approx. 100 μM. The inhibitors of Irp9, with varied substitution at C-2 and C-4, had inhibition constants ranging from 19 μM to 1110 μM. A homology model of Irp9 with bound inhibitor in a conformation suitable to fit into the presumed active site was constructed. This model presents a good template for site-directed mutagenesis studies, which will give further clues to elucidate the steps in the Irp9 catalytic sequence.
Despite the significant developments in mechanistic studies of the chorismate-utilizing enzymes anthranilate synthase, ADC synthase and isochorismate synthase, there are still many details that remain to be determined. It is not known how each enzyme controls the nature of the nucleophile, and the position of the chorismate ring at which nucleophilic attack occurs. ADC synthase and anthranilate synthase must both have a mechanism for preventing water acting as a nucleophile, as it does in isochorismate synthase, and for directing the attack of ammonia at C-2 and C-4 respectively.
The reason that anthranilate synthase and salicylate synthase catalyse a pyruvate lyase reaction, whereas isochorismate synthase and ADC synthase do not, is also not understood. Future mutagenesis and structural studies will hopefully reveal the basis for the interesting structure–function relationships in this family of enzymes. From an evolutionary point of view, comparing these enzymes will also help us to understand their mono- or bi-functionality.
Coenzymology: the biochemistry of vitamin biogenesis and cofactor-containing enzymes: Independent Meeting held at King's College, Cambridge, U.K., 4–7 April 2005. Organized and Edited by A.G. Smith (Cambridge, U.K.) and A.W. Munro (Leicester, U.K.).
Abbreviations: ADC, 4-amino-4-deoxychorismate
- © 2005 The Biochemical Society