2.3- DHBA

The CFF-mediated conversion of salicylate to 2,5- and 2,3-DHBA and catechol was used as a semi-quantitative assay to determine the levels of redox-active Fe/Cu, as follows in 100 pi reaction mixture, containing 50 pi CFFs and salicylate and ascorbate (1 mM, each) in KH buffer was incubated for Ih at 37 C. To terminate the incubation, ice-cold TCA (3% final concentration) was added, and the suspensions were centrifuged at 12,000g for 1 min. The supernatant was analyzed for 2,5- and 2,3-DHBA and catechol by HPLC coupled to an electrochemical detector (HPLC-ECD), as previously described [10]. 

As shown in Fig. 3, 2,3-DHBA and 2,5-DHBA may derive from SA as the products of metabolic inactivation which arises from additional hydroxylation of the aromatic ring. The rapid conjugation of SA with glucose to form P-D-o-glucosylsalicylic acid is the main metabolic inactivation mechanism in tobacco [37,38]. The glucose ester of SA was detected in soybean cell cultures fed with [ C]-labelled SA [39] and a small amount of these compound is also detected in tobacco [38]. 

In cell cultures of Catharanthus roseus 2,3-DHBA was found to be produced after elicitation of the cells with fungal cell-wall preparations [40,41]. The production of this compound in cell cultures was paralleled by an increase in the activity of the enzyme isochorismate synthase [40]. Although direct evidence for the intermediacy of iso-chorismate is not yet reported, it seems that the isochorismate biosynthetic pathway for benzoic acid derivatives can also be found in plants. 

Isochorismate synthase, that might be involved in the biosynthesis of 2,3-DHBA, has recently been purified from Catharanthus roseus cell-suspension cultures and subsequently its gene was cloned (L. van Tegelen, P. Moreno, A. Croes, G. Wullems and R. Verpoorte, submitted for publication). Two isoforms of the enzyme were purified and characterized. Both have an apparent molecular mass of 65 kD. The Km values for chorismic acid are 558 pM and 319 p.M for isoform I and II respectively. The enzymes are not inhibited by aromatic amino acids and require Mg for enzyme activity. The isolated cDNA encodes a protein of 64 kD with a A-terminal chloroplast targeting signal. The deduced amino acid sequence shares homology with bacterial isochorismate synthases, and also with anthranilate synthases, another chorismate utilizing enzyme. 

This pathway was first found in microorganisms which produce SA or the related compound 2,3-DHBA. The function of these compounds is different from that in plants. Under aerobic growth conditions, iron occurs in the environment as the highly insoluble Fe(OH)j. To overcome the problem of Fe " deficiency almost all bacteria and fungi have evolved high-affinity Fe transport systems based on the synthesis of low-molecular-mass... 

The phenolates are based on the iron binding capacity of 2,3-DHBA or SA. These siderophores range in structure from the free monomer 2,3-DHBA or SA which form Fe(2,3-DHBA)3 or Fe(SA)3 complexes [61-64], to single amino acid conjugates with for instance serine, glycine, cysteine, and lysine [55,65-70], and even more complex molecules like enterobactin (cyclic triester of 2,3-DHBA-serine) [71-73]. 

Thus various microorganisms are able to biosynthesize SA and 2,3-DHBA in considerable amounts. In connection with the biosynthesis of siderophores during Fe deficiency, SA and 2,3-DHBA are synthesized [74,64]. 

DHBA, followed by a dehydration. Such a pathway would require a co-factor as NAD, similar to the 2,3-DHBA biosynthesis. However, no evidence for such a NAD dependent conversion could be found in M smegmatis [77,78]. SA can also be obtained as an intermediate in naphthalene degradation [81]. 

In 1967 it was shown by Young et al. [84] that 2,3-DHBA in K. pneumoniae and E. coli is produced via the shikimic acid pathway (Fig. 1). Evidence showed that the centra) intermediate chorismic acid, leading to the aromatic amino acid pathways, is also the precursor for 2,3-DHBA. The formation of 2,3-DHBA required NAD, and Mg (Fig. 4)... 

The first intermediate in the biosynthesis of 2,3-DHBA in K. pneumoniae is isochorismic acid. The conversion of chorismic acid to isochorismic acid requires Mg ... 

Experiments on the rate of 2,3-DHBA formation from chorismic acid and the observation that chorismic acid, in the absence of NAD was converted to a compound which could serve as a substrate for 2,3-DHBA formation, indicated that at least two steps were concerned in the conversion of chorismic acid to 2,3-DHBA [84,86]. The first intermediate is isochorismic acid, which is converted to the second intermediate... 

Young, Batterham and Gibson [87] firmly established isochorismate as an intermediate in 2,3-DHBA biosynthesis. Isochorismic acid is an unstable compound and at room temperature, in aqueous solution at pH 7, it decomposes readily to a mixture of SA and... 

DHBA is also an intermediate in the catabolism of L-tryptophan. 2,3-DHBA is formed in this pathway from anthranilate, by the enzyme anthranilate hydroxylase through deamination [89]. 

Enzymes and genes involved in the 2,3-DHBA biosynthesis When in 1968 the intermediates of 2,3-DHBA biosynthesis were identified [85], it became clear that isochorismate synthase (isochorismate hydroxy mutase) is the enzyme that converts chorismic acid to isochorismic acid. 2,3-Dihydro-2,3-DHBA synthase (or isochorismatase) converts isochorismic acid to 2,3-dihydro-2,3-DHBA and finally... 

Siderophores are produced by microorganisms to overcome the risk of Fe deficiency. The biosynthesis of siderophores and the intermediates SA or 2,3-DHBA is therefore dependent on Fe availability [57,68,74]. The production of 2,3-DHBA-glycine in B. subtilis is inversely proportional to the amount of iron in the culture [117]. When iron is available the Fe(2,3-DHBA)3 complex is involved in the control of the biosynthesis [117]. The production of mycobactin is repressed by high levels of Fe or Fe and Zir, SA concentration increased both when Fe and Fe were omitted from the medium [118]. The biosynthesis of pyochelin can also be reduced by other transition metals like Co, Mo , Ni ", and Cu [82] It seemed that iron inhibits the activities of the enzymes required for the biosynthesis of SA or 2,3-DHBA [78,119]. Iron is not an inhibitor of the activity of the enzymes themselves, but it influences the synthesis of the enzymes [120,121]. 

E. coli there is a transcriptional linkage of the 2,3-DHBA gene cluster. Control sequences directing iron-regulated co-transcription of the enterobactin biosynthesis genes were... 

When a P. aeruginosa mutant (PALS 128) was grown under iron rich conditions, the specific activity of the SA-forming enzymes was below the limits of detection [79]. Liu et al. [88], suggest that entC gene expression may be limited at the translational level as well, even when the operon is induced under iron deficiency. This may be understandable because chorismic acid is an essential metabolite for Phe, Trp, Tyr, folate and ubiquinone synthesis. In B. subtilis it was shown that the accumulation of 2,3-DHBA(Glycine) was influenced by the levels of aromatic amino acids and anthranilic acid. Anthranilic acid inhibited the synthesis of DHBA from chorismic acid [117]. It seemed that the reduction in phenolic acid accumulation caused by aromatic amino acids is a consequence of enzyme repression [121]. The synthesis of 2,3-DHBA in B. subtilis is also reduced by other phenolic acids, such as m-substimted benzoic acids. Inhibition of accumulation of phenolic acid by other phenolic acids, would indicate a fairly specific effect on phenolic acid synthesis, but not on the accumulation of coproporphyrin that also accumulates in iron-deficient cultures oiB. subtilis [121]. 

A feedback inhibition has been detected in B. subtilis, using the ferrisiderophore reductase. This enzyme reduces iron from the ferrisiderophore. The rate at which the ferrisiderophore reductase reduces iron from ferrisiderophores may signal the aromatic pathway about the demand for chorismic acid for 2,3-DHBA synthesis [128,129]. The reductase may have a regulatory effect on chorismate synthase activity. Chorismate synthase may have oxidizable sulfhydryl groups that, when oxidized, may slow the synthesis of chorismic acid [128-130]. There seemed to be no repression or inhibitory effect of 2,3-DHBA or SA on its own biosynthesis [78,121]. Also the endproduct mycobactin (sole endproduct) does not inhibit SA biosynthesis [78]. 

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