DAHP synthase

Multi-enzymatic scheme for synthesis of 3-deoxy-arab/no-heptulosonic acid 7-phosphate based on the catalysis of DAHP synthase, and products generated consecutively in vivo by advanced microbial pathw/ay engineering. 

An aldolase specific for cleavage of 2-keto-3-deoxy-6-phospho-o-gluconate (35) (KDPGlc aldolase or KdgA EG 4.1.2.14) is produced by many species of bacteria for degradation of 6-phosphogluconate to give pyruvate and d- 

Stereospecific conversion of 3-fluoro-labeled PEP analogs to substituted DAMP derivatives by use of DAMP synthase. 

Aldol reactions catalyzed in vivo by the 2-keto-3-deoxy-6-phospho-D-gluconate and 2-keto-3-deoxy-6-phospho-D-galactonate aldolases. 

---

Figure 1. Hypothetical mechanism for shuttling of intermediates of the common aromatic pathway between plastidic and cytosolic compartments. Enzymes denoted with an asterisk (DAHP synthase-Co, chorismate mutase-2, and cytosolic anthranilate synthase) have been demonstrated to be isozymes located in the cytosol. DAHP molecules from the cytosol are shown to be shuttled into the plastid compartment in exchange for EPSP molecules synthesized within the plastid. Abbreviations C3, phosphoenolpyruvate C4, erythrose 4-P DAHP, 3-deoxy-D-arabino-heptulosonate 7-phosphate EPSP, 5-enolpyruvylshikimate 3-phosphate CHA, chorismate ANT, anthranilate TRP, L-tryptophan PPA, prephenate AGN, L-arogenate TYR, L-tyrosine and PHE, L-phenylalanine.

Of the separately compartmented isozyme pairs that exist for DAHP synthase, chorismate mutase, and anthranilate synthase, each isozyme member of a given pair has different properties of regulation and other distinctive characteristics (see Tables I and II). This suggests a high probability that each isozyme is the gene product of a different gene. 

Table I. Differential Properties of DAHP Synthase Isozymes...

Assay at low pH (7.0) can be used to increase the selectivity of assay conditions for DAHP synthase-Mn in isozyme mixtures. 

Figure 4. Reactions catalyzed by the broad-specificity DAHP synthase-Co of higher plant cytosol. Condensation of PEP and erythrose 4-P (top) yields 3-deoxy-D-arabino-heptulosonate 7-P (DAHP), whereas condensation of PEP and D-glyceraldehyde 3-P (G-3-P) yields 2-keto-3-deoxy-D-threo-hexulosonate 6-P (DTHP).

Figure 6. Effects of various treatments or manipulations upon levels of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), and the separately compartmented isozymes of DAHP synthase and chorismate mu-tase. Upwardly pointed arrows indicate a positive response (enzyme elevation) to the indicated treatment, whereas horizontal arrows indicate no change in enzyme level. References documenting the results shown are line 1 (54 our results with DAHP synthase and chorismate mutase isozymes) line 2 (49,55) line 3 (49,50,56) line 4 (57) line 5 (51) line 6 (52).

We have examined the time course of changes induced in isozymes of chorismate mutase and DAHP synthase in potato tubers following mechanical wounding (Table III). In each case both isozymes responded—the plastidic isozyme responding sooner and to a greater extent than the cytosolic isozyme. All five of the other pathway enzymes so far examined were induced by mechanical wounding. 

Accordingly, a strain of E. coli was first engineered to produce elevated levels of DHQ by increasing the levels of certain key enzymes transketolase, 3-deoxy-D-arabino-heptulosonic acid 7-phospate (DAHP) synthase, and DHQ synthase. Also, the strain has reduced levels of DHQ dehydratase, which if present would divert some of the metabolic flow into the biosynthesis of aromatic amino acids its blockage results in higher production of quinic acid. 

Most bacteria and fungi have three isozymes of DAHP synthase, each controlled by feedback inhibition by one of the three products tyrosine, phenylalanine, or tryptophan. In E. coli these are encoded by genes aro F, am G, and aro H, respectively.11-123 All of the enzymes contain one atom of iron per molecule and show spectral similarities to hemerythrin.13... 

The product of the DAHP synthase, 3-deoxy-D-arabino-heptulosonate 7-phosphate, is shown in its cyclic hemiacetal form at the beginning of Eq. 25-2. Its conversion to 3-dehydroquinate is a multistep process that is catalyzed by a single enzyme, 14/15 which is the product of E. coli gene am B. The elimination of... 

The six carbons of the benzene ring of the aromatic amino acids are derived from the four carbons of erythrose 4-phosphate and two of the three carbons of phosphoenolpyruvate (PEP). The initial step in the pathway (Fig. 25-1, step a) is the condensation of erythrose 4-P with PEP and is catalyzed by 3-deoxy-D-arafrmo-heptulosonate-7-phosphate (DAHP) synthase. Closely analogous to an aldol condensation, the mechanism provides a surprise.10 When PEP containing lsO in the oxygen bridge to the phospho group reacts, the lsO is retained in the eliminated phosphate biochemical intuition would suggest that it should stay in the... 

The shikimate pathway is common to both plants and microorganisms (Figure 3-3). Shikimate is synthesized from the substrates phosphoewo/pyruvate (3.9) and erythrose 4-phosphate (3.17). These two precursors are derived from glycolysis and the pentose phosphate pathway, respectively, and are condensed to 3-deoxy-D-ara6/ o-heptulosonate 7-phosphate (DAHP 3.18) by the enzyme DAHP synthase. The subsequent steps result in the formation of 3-dehydro-quinate (3.19) by the enzyme 3-dehydroquinate synthase, 3-dehydroshikimate... 

By use of synthetic medium the formation of 3,4-trans-CH D was maintained for a 36-h cultivation period, resulting in accumulation of up to 790 mg L 1 3,4-trans-CHD 1 [11]. With this strain, however, it is necessary to separate the growth and production phases, because the substances for which E. coli strain BN117 is known to be auxotrophic, i.e. tryptophan, tyrosine, phenylalanine, proline, arginine, histidine, and p-aminobenzoate, also partially inhibit entry to the chorismate biosynthesis pathway (feedback inhibition of DAHP-synthase). 

For the aromatic pathway (Figure 30.20), the critical control points are the condensation of phosphoenolpyruvate and erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonate 7-phosphate, DAHP, by DAHP synthase. For tryptophan, the formation of anthranilic acid from chorismic acid by anthranilate synthase is the second critical control point. The transcriptional regulation was overcome through the use of alternative promoters and allosteric regulation was circumvented by the classical technique of selection for feedback-resistant mutants using toxic analogues of the repressing compounds. 

In a very imaginative piece of research Frost and coworkers have developed a plasmid-based method for synthesizing aromatic amino acids, by incorporating the genes that code for the enzymes that perform the series of conversions from D-fructose-6-phosphate to D-erythrose-4-phosphate to 3-deoxy-D-arabinoheptulosonic acid-7-phos-phate (DAHP) near each other on a plasmid that can be transformed in E. coli. The enzymes are the thiamin diphosphate-dependent enzyme transketolase in the nonoxida-tive pentose shunt and DAHP synthase. The DAHP is then converted to the cyclic dehydroquinate, a precursor to all aromatic amino acids L-Tyr, L-Phe and L-Trp165,166 (equation 27). 

DAHP synthase combines E4P and PEP, which are both derived from the central metabolism (see Figs. 8.4 and 8.5 for more details). The biosynthesis of... 

C. glutamicum and E. coli, which share very similar biosynthetic pathways and control architectures, have been subjected to pathway engineering for the production of L-Trp. Modifications that have been reported include the, now familiar, feedback-resistant DAHP synthase and the enzymes in the Trp pathway were freed from regulation [72, 97] and overexpressed. 

---