Enzyme DAHPS

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... 

Comprehensive studies are now required, however, to determine the feedback properties of all of the enzymes leading to Phe (1)/Tyr (2) biosynthesis, including the individual Arabidopsis ADTs and the yet to be provisionally identified aminotransferases both in vivo and in vitro. This is a particularly interesting and important question, considering that feedback-insensitive isoforms exist for the other branch point enzymes DAHP synthase, CM, and AS. 

The enzyme DAHP synthase regulates the carbon flow in the shikimate pathway. Different biotic and abiotic stresses, including mechanical wounding and fungal elicitation, induce the accumulation of DAHP synthase mRNA, and, therefore, of phenolic metabolites. 

Studies on the regulation of the common pathway of aromatic biosynthesis in several micro-organisms have shown that control of the first reaction (Figure 1.2), the conversion of o-erythrose-4-phosphate (7) and phosphoenolpyruvate (8) to 3-deoxy-o-arabino-heptulosonic acid-7-phosphate (9, DAHP), catalysed by the enzyme DAHP synthetase (EC 4.1.2.15) is an important factor in the overall control of the pathway In a number of enteric bacteria this enzyme exists in multiple molecular forms each of which is under the feedback control of a specific end-product. Thus in Escherichia coli there are three DAHP synthetases (iso-enzymes), the activity and formation of which are controlled by the three aromatic amino acids The formation and activity of DAHP synthetase... 

Deoxy-D-araZu o-heptulosonate-7-phosphate synthetase (DAHPS) is the initial enzyme in the pathway responsible for the synthesis of aromatic compounds in microorganisms and plants. It catalyses the reaction shown in Equation (4) ... 

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.

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 shikimate pathway begins with a coupling of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to give the seven-carbon 3-deoxy-D-araWno-heptulosonic acid 7-phosphate (DAHP) (Figure 4.1). This reaction, shown here as an aldol-type condensation, is known to be mechanistically more complex in the enzyme-catalysed version several of the other transformations in the pathway have also been found to be surprisingly... 

Fourthly, biotransformations have been used for the synthesis of 3-deoxy-2-glyculosonic acids, using whole cells or purified enzymes. For instance, 3-deoxy-D-araZu rao-heptulosonic acid (DAH) and its 7-phosphate (DAHP, 122) have been produced directly from D-glucose by mutants of E. coli JB-5, that lack dehydroquinate synthase, the enzyme that converts DAHP into the cyclic intermediate dehydroquinic acid (DHQ, Scheme 14). Both DAH and DAHP are secreted into the medium. The dephosphorylated product could be generated in vivo by a phosphatase acting on DAHP.312... 

Synthesis using immobilized enzymes has also been applied to the preparation of DAHP.320 The 7-phosphonate of DAH, having a C P... 

Wild apricot (Prunus armenica L.) grows naturally in hilly areas of northern India. It is highly acidic, fibrous, and low in TSS, and, thus, not utilized commercially. Preparation and evaluation of a vermouth from its fruit was undertaken (Abrol, 2009). Vermouths at different sugar (8,10, and 12 °Brix), alcohol (15%, 17%, and 19%), and spices levels (2.5% and 5%) were prepared. Those used in extract preparation are shown in Plate 8.1. The base wine was prepared from crushed fruit, adjusted to 24 °Brix, and diluted in a 1 2 ratio with water. To this mixture was added 200 ppm sulfur dioxide, 0.1% diammonium hydrogen phosphate (DAHP), and 0.5% pectinase enzyme. A 24-h active yeast culture initiated fermentation. The procedure is illustrated in Fig. 8.4. A maturation period of 6 months improved the quality of the vermouth. 

Other aldolases have been described that do not rely on DAHP as a substrate. One such enzyme, 2-deoxyribose-5-phosphate aldolase (E.C. 4.1.2.4), has been cloned into E. coli, accepts a broad range of donor and acceptor aldehydes, and has been used to synthesize a number of heterocycles having utility as epithiolone synthons.259... 

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). 

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. 

Scheme 5.40. Preparative scale production of DAHP by multi-enzyme synthesis. Ei = hexoki-nase, E2 = pyruvate kinase, E3 = transketolase + D-ribose 5-P, E4 = DHAP synthetase. P, = inorganic phosphate, P = PC>32. ...

DAHP synthetase, 251, 255 Dehydroquinase, 258 Dehydroshikimate riductase, 259 Dextrans, 341 acid hydrolysis of, 349 chain lengths of, 345, 346 cuprammonium complexes of, 355 electron microscope studies on, 349 enzymic synthesis of, 342, 345, 355 from sucrose, 342 flow birefringence studies on, 349 fructose-containing, 359 infrared absorption spectra of, 352 isolation of, 343... 

Fig. 12. Metabolic pathways leading to the biosynthesis of phenylalanine, anthranilic acid, and cyclopenin in Penicillium cyclopium (67). Symbols in parenthesis indicate observed feedback inhibition (-) or activation (-I-) of enzyme activities by L-amino acids. Number in circles (pkat/cm of mycelial area) represent either in vitro activities of the enzymes indicated or the rate of alkaloid formation in vivo. All data were measured after 7 days of growth in surface cultures. E 4-P, Erythrose 4-phosphate PEP, phosphoenolpyruvate DAHP, 3-deoxo-D-arabiuoheptuIosonic acid-7-phosphate InGP, inositol glycerophosphate SAM, 5-adeno ylmethionine.

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