Phenylpyruvate route, phenylalanine

Figure 2. Alternative enzymatic routing for L-phenylalanine biosynthesis. Dehydration followed by transamination defines the phenylpyruvate route, whereas the reverse order of reactions defines the arogenate route. Abbreviations GLU, L-glutamate aKG, 2-ketoglutarate.

A much more serious genetic disease, first described by Foiling in 1934, is phenylketonuria. Here the disturbance in phenylalanine metabolism is due to an autosomal recessive deficiency in liver phenylalanine hydroxylase (Jervis, 1954) which normally converts significant amounts of phenylalanine to tyrosine. Phenylalanine can therefore only be metabolized to phenylpyruvate and other derivatives, a route which is inadequate to dispose of all the phenylalanine in the diet. The amino acid and phenylpyruvate therefore accummulate. The condition is characterized by serious mental retardation, for reasons which are unknown. By the early 1950s it was found that if the condition is diagnosed at birth and amounts of phenylalanine in the diet immediately and permamently reduced, mental retardation can be minimized. The defect is shown only in liver and is not detectable in amniotic fluid cells nor in fibroblasts. A very sensitive bacterial assay has therefore been developed for routine screening of phenylalanine levels in body fluids in newborn babies. 

Although 2-phenylethanol can be synthesised by normal microbial metabolism, the final concentrations in the culture broth of selected microorganisms generally remain very low [110, 111] therefore, de novo synthesis cannot be a strategy for an economically viable bioprocesses. Nevertheless, the microbial production of 2-phenylethanol can be greatly increased by adding the amino acid L-phenylalanine to the medium. The commonly accepted route from l-phenylalanine to 2-phenylethanol in yeasts is by transamination of the amino acid to phenylpyruvate, decarboxylation to phenylacetaldehyde and reduction to the alcohol, first described by Ehrlich [112] and named after him (Scheme 23.8). 

In the mid to late 1980s, many research groups focused on methods and processes to prepare L-phenylalanine (Chapter 3). This was a direct result of the demand for the synthetic, artificial sweetener aspartame. One of the many routes studied was the use of phenylalanine dH (Scheme 19.4, R = C6H5CH2) with phenylpyruvate (PPA) as substrate.57-58 This enzyme from Bacillus sphaericus shows a broad substrate specificity and, thus, has been used to prepare a number of derivatives of L-phenylalanine.59 A phenylalanine dH isolated from a Rhodococcus strain M4 has been used to make L-homophenylalanine (.S )-2-amino-4-pheny I butanoic acid], a key, chiral component in many angiotensin-converting enzyme (ACE) inhibitors.40 More recently, that same phenylalanine dH has been used to synthesize a number of other unnatural amino acids (UAAs) that do not contain an aromatic sidechain.43... 

Fig. 4-2. Simplified reaction route illustrating the formation of lignin precursors. 1, 5-Dehydroquinic acid 2, shikimic acid 3, phenylpyruvic acid 4, phenylalanine 5, cinnamic acid 6, ferulic acid (Ri=H and R2=OCH3), sinapic acid (R,= R2=OCH3), and p-coumaric acid (R1=R2 = H) 7, coniferyl alcohol (Ri = H and R2=OCH3), sinapyl alcohol (Rj = R2=OCH3), and p-coumaryl alcohol (R =R2=H) 8, the corresponding glucosides of 7.

In Thaurea aromatica, phenylalanine was found to be converted to benzoyl-CoA by an anaerobic a-oxidation route via phenylpyruvate and phenylacetate [route 4 on Figure 3, (iP)]. Further, a non-oxi tive, retro-aldol route to benzoyl-CoA via benzaldehyde was also described in plants (route S on Figure 3). Feeding these intermediates to our engineered S. lividans strains did not lead to the production of soraphen A (Table I), indicating that neither of these two metabolic routes are operational in this host under the tested conditions. 

Figure 15.3-5. Enzymatic routes to L-phenylalanine via phenylpyruvate191. (i) Reductive animation of phenylpyruvate by PheDH with simultaneous NADH regeneration using FDH.

Classical phenylketonuria is an hereditary defect in the synthesis of Phe hydroxylase (the enzyme may be absent or inactive), which affects about 1 infant in 10,000. These individuals are unable to convert Phe into tyrosine, and the major route of Phe metabolism is thus blocked. Phenylpyruvate and phenylacetic acid are excreted in the urine. The condition is accompanied by defective pigmentation and, if untreated, by severe mental retardation (hence the other name, phenylpyruvic oligophrenia, also known as Polling s syndrome). Tlie urine of newborn infants is now routinely tested (Guthrie test) for the presence of phenylketones the condition can be compensated by a diet low in phenylalanine, and the typic mental retardation is thereby avoided. Other types of phenylketonuria are due to defective reduction or synthesis of dihydrobiopterin (see Inborn errors of metabolism). 

Bacteria, fungi, and plants share a common pathway for the biosynthesis of aromatic amino acids with shikimic acid as a common intermediate and therefore named after it—the shikimate pathway. Availability of shikimic acid has proven to provide growth requirements to tryptophan, tyrosine, and phenylalanine triple auxotrophic bacterial strains. Chorismate is also the last common precursor in the aromatic amino acid biosynthetic pathway, but the pathway is not named after it, as it failed to provide growth requirements to the triple auxotrophs. The aromatic biosynthetic pathway starts with two molecules of phosphoenol pyruvate and one molecule of erythrose 4-phosphate and reach the common precursor, chorismate through shikimate. From chorismate, the pathway branches to form phenylalanine and tyrosine in one and tryptophan in another. Tryptophan biosynthesis proceeds from chorismate in five steps in all organisms. Phenylalanine and tyrosine can be produced by either or both of the two biosynthetic routes. So phenylalanine can be synthesized from arogenate or phenylpyruvate whereas tyrosine can be synthesized from arogenate or 4-hydroxy phenylpyruvate. 

Fig. 2. Flow routes to phenylalanine and tyrosine in nature. Abbreviations CHA, chorismate PPA, prephenate HPP, 4-hydroxyphenylpyruvate AGN, L-arogenate PPY, phenylpyruvate TYR, L-tyrosine PHE, L-phenylalanine.

In higher plants prephenate dehydratase, which converts prephenate to phenylpyruvate, has never been demonstrated. Since we have recently demonstrated the presence of arogenate dehydratase, which converts -arogenate to L-phenylalanine, in several higher plants (this paper), the arogenate route to phenylalanine may be characteristic of higher plants. 

Although the pulvinic acid amide derivatives rhizocarpic acid and epanorin (58) could originate from simple coupling between the appropriate amino acid and the pulvinic acid, Maass (66) has proposed a direct route to pulvinamide itself (viz 58, R = H), involving condensation between phenylalanine and phenylpyruvic acid. 

Experiments with (2S,3R)- and (2/, 35 )-[3- H]phenylalanines gave tritium retentions of 44% and 24%, respectively (Vederas and Tamm, 1976). Simultaneous incorporation of equal amounts of both enantiomers led to the expected 34% retention of hydrogen label. Transamination occurs stereo-specifically at position 2 of the amino acid therefore, the participation of at least two enzymes with different stereochemical requirements at the 3 position is reasonable. Two biosynthetic pathways are consistent with the data available (see Fig. 13). Path A in Fig. 13 depicts (2 S )-phenylalanine as the actual precursor which is in rapid equilibrium with its enantiomer in path B phenylpyruvic acid (49), derived directly from shikimic acid, is the primary precursor. Considerable suppression of the incorporation of D-amino acid by phenylpyruvic acid (49) indicated that the naturally abundant L-enantiomer is the actual primary precursor, thus demonstrating that path A (Fig. 13) is probably the main biosynthetic route. Both enantiomers are in rapid equilibrium with phenylpyruvic acid (49) via the action of aminotransferases or amino acid oxidases. The stereochemistry of hydrogen loss... 

This route, often called the shikimic acid pathway involves the condensation of phosphoenolpyruvate (2) and a 4-carbon sugar erythrose-4-phosphate (1) which is derived from the pentose phosphate pathway. The product of this reaction is converted to shikimic acid (3). Phosphorylation of shikimic acid to yield 5-phosphoshikimic acid (4) is followed by the addition of another molecule of phospho-enol pyruvate (2) which results in the synthesis of prephenic acid (5). Aromatization of the prephenic acid can give rise to phenylpyruvic acid (6) which upon transamination becomes phenylalanine. The carbon skeletons of the other aromatic amino acids, tryptophane and tyrosine are also synthesised via the shikimic acid pathway as is lignin and many of the aromatic secondary products described in Chapter 6. 

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