Genetics and biochemistry of the phenylketonuria-present state

Summary Phenylketonuria is an autosomal recessive inherited disease caused by a disturbance in the phenylalanine hydroxylating system. Phenylalanine is converted to tyrosine by phenylalanine hydroxylase, which is located mainly in the liver. This enzyme needs the reduced cofactor tetrahydrobiopterin to be active. In phenylketonuria, low or zero enzyme activity is measured. Enzyme activity higher than 5% compared with that in normal controls is correlated to hyperphenylalaninemia. Dihydropteridine reductase regenerates the active cofactor. A defect in this enzyme or in the biosynthesis of the cofactor results in phenylketonuria which does not respond to dietary treatment because the biosynthesis of neurotransmitters is impaired.     

Evaluation of the phenylalanine tolerance test in detection of heterozygote carriers of phenylketonuria gene]

The study aimed at identifying heterozygotic phenylketonuria gene carriers with phenylalanine tolerance test performed in Lublin region. Serum phenylalanine concentration has been assayed during fasting and 1 and 2 hours following oral phenylalanine load in the dose of 0.1 g per 1 kg body weight. The study involved 203 individuals of the general population and 29 heterozygotes with phenylketonuria gene. Blood serum phenylalanine was assayed with Guthrie' technique. Statistical analysis has shown that hyperphenylalaninemia is relatively frequent in fasting individuals of the general population (59.1%). The same was demonstrated in 5 heterozygotes. Phenylalanine tolerance test did not allow to identify heterozygotic carries of phenylketonuria gene in the general population though fasting and after phenylalanine load increased blood serum levels of this amino acid are a criterium of hyperphenylalaninemia in the group of tested individuals (29%).     

The PKU locus in man is on chromosome 12

Classical phenylketonuria (PKU) is a typical example of inborn errors in metabolism and is characterized by a complete lack of the hepatic enzyme phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine. The genetic disorder causes impairment of postnatal brain development, resulting in severe mental retardation in untreated children. The disease is transmitted as an autosomal recessive trait and has a collective prevalence of about one in 10,000 among Caucasians, so that 2% of the population are carriers of the PKU trait. We have recently reported the cloning of human phenylalanine hydroxylase cDNA and that the human chromosomal phenylalanine hydroxylase gene is encoded by a unique DNA sequence. Using the human phenylalanine hydroxylase cDNA clone to analyze a clonal human/mouse hybrid cell panel by Southern hybridization, the phenylalanine hydroxylase gene has been assigned to human chromosome 12. Since the hypothesis that classical PKU is caused by structural mutations in the phenylalanine hydroxylase gene itself rather than through some transregulatory mechanisms has recently been confirmed by gene mapping, the PKU locus in man is determined to be on chromosome 12.     

Novel photoreactive cinnamic acid analogues to elucidate phenylalanine ammonia-lyase

4-[3-(Trifluoromethyl) diazirinyl] cinnamic acid derivatives were synthesized to elucidate properties of phenylalanine ammonia-lyase (PAL). 2-Methoxy and 2-biotinylated alkoxy compounds have inhibitory activity on the formation of phenylalanine from cinnamic acid. Specific photolabeling of the enzyme was detected using biotinylated derivatives without the use of radioisotopes. The results indicated that the 4-[3-(trifluoromethyl) diazirinyl] skeleton will be a suitable photoreactive compound to elucidate regulation of phenylpropanoid biosynthesis.     

Production of plant-specific flavanones by Escherichia coli containing an artificial gene cluster

In plants, chalcones are precursors for a large number of flavonoid-derived plant natural products and are converted to flavanones by chalcone isomerase or nonenzymatically. Chalcones are synthesized from tyrosine and phenylalanine via the phenylpropanoid pathway involving phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL), and chalcone synthase (CHS). For the purpose of production of flavanones in Escherichia coli, three sets of an artificial gene cluster which contained three genes of heterologous origins--PAL from the yeast Rhodotorula rubra, 4CL from the actinomycete Streptomyces coelicolor A3(2), and CHS from the licorice plant Glycyrrhiza echinata--were constructed. The constructions of the three sets were done as follows: (i) PAL, 4CL, and CHS were placed in that order under the control of the T7 promoter (P(T7)) and the ribosome-binding sequence (RBS) in the pET vector, where the initiation codons of 4CL and CHS were overlapped with the termination codons of the preceding genes; (ii) the three genes were transcribed by a single P(T7) in front of PAL, and each of the three contained the RBS at appropriate positions; and (iii) all three genes contained both P(T7) and the RBS. These pathways bypassed C4H, a cytochrome P-450 hydroxylase, because the bacterial 4CL enzyme ligated coenzyme A to both cinnamic acid and 4-coumaric acid. E. coli cells containing the gene clusters produced two flavanones, pinocembrin from phenylalanine and naringenin from tyrosine, in addition to their precursors, cinnamic acid and 4-coumaric acid. Of the three sets, the third gene cluster conferred on the host the highest ability to produce the flavanones. This is a new metabolic engineering technique for the production in bacteria of a variety of compounds of plant and animal origin.     

Ammonia lyases and aminomutases as biocatalysts for the synthesis of α-amino and β-amino acids

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.     

Genetics of the mammalian phenylalanine hydroxylase system. Studies of human liver phenylalanine hydroxylase subunit structure and of mutations in phenylketonuria.

Phenylalanine hydroxylase was purified from crude extracts of human livers which show enzyme activity by usine two different methods: (a) affinity chromatography and (b) immunoprecipitation with an antiserum against highly purified monkey liver phenylalanine hydroxylase. Purified human liver phenylalanine hydroxylase has an estimated mol. wt. of 275 000, and subunit mol. wts. of approx. 50 000 and 49 000. These two molecular-weight forms are designated H and L subunits. On two-dimensional polyacrylamide gel under dissociating conditions, enzyme purified by the two methods revealed at least six subunit species, which were resolved into two size classes. Two of these species have a molecular weight corresponding to that of the H subunit, whereas the other four have a molecular weight corresponding to that of the L subunit. This evidence indicates that active phenylalanine hydroxylase purified from human liver is composed of a mixture of sununits which are different in charge and size. None of the subunit species could be detected in crude extracts of livers from two patients with classical phenylketonuria by either the affinity or the immunoprecipitation method. However, they were present in liver from a patient with malignant hyperphenylalaninaemia with normal activity of dihydropteridine reductase.     

Two mutations within the coding sequence of the phenylalanine hydroxylase gene

Summary Two previously unidentified mutations at the phenylalanine hydroxylase locus were found during a study of the relationship between genotype and phenotype in phenylketonuria and hyperphenylalaninemia. One mutation eliminates the BamHI site in exon 7 and the other eliminates the HindIII site in exon 11 of the phenylalanine hydroxylase gene. They were suspected because of deviating restriction fragment patterns and confirmed by amplification, via the polymerase chain reaction, of exon 7 and exon 11, respectively, followed by digestion with the appropriate restriction enzyme. Direct sequencing of amplified mutant exon 7 revealed a G/C to T/A transversion at the first base of codon 272, substituting a GGA glycine codon for a UGA stop codon. Direct sequencing of amplified mutant exon 11 revealed a deletion of codon 364, a CTT leucine codon. The exon 7 mutation can be expected to result in a truncated protein and the exon 11 mutation in the elimination of an amino acid in the catalytic region of the enzyme. A patient who is a compound heterozygote for these two mutations has classical phenylketonuria. It is concluded that each of the two mutations leads to a profound loss of enzymatic activity. The segregation of these mutations with disease alleles in 4 and 2 families, respectively, supports the hypothesis that multiple mutations at the phenylalanine hydroxylase locus explain the variable phenylalanine tolerance in patients with phenylalanine hydroxylase deficiency.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

Cinnamic acid intermediates as precursors to mesembrine and some observations on the late stages in the biosynthesis of the mesembrine alkaloids

Experiments with various labelled cinnamic acid derivatives establish, in conjunction with previous work, that the incorporation of phenylalanine into the 3a-aryl octahydroindole ring system of the mesembrine alkaloids occurs via the intermediacy of cinnamic acid and 4′-hydroxycinnamic acid. The major pathway to the 3′,4′-di-oxyaryl substituted alkaloids proceeds via 4′-hydroxydihydrocinnamic acid (4′-phloretic acid), and 3′4′-dioxy-genated cinnamic acids are not involved as intermediates on this major pathway. In accord with this latter finding, the 3′-aryl oxygen substituent is introduced at a late state in the biosynthesis as evidenced by the bioconversion in S. strictum of sceletenone to mesembrenol and other related alkaloids. The late stages in the biosynthesis of the alkaloids are shown to involve the sequence: sceletenone, 4′-O-demethylmesembrenone, mesembrenone. Mesembrenone is converted to mesembrine, mesembrenol and mesembranol.     

The regulation of phenylalanine hydroxylase in rat tissues in vivo. The maintenance of high plasma phenylalanine concentrations in suckling rats: a model for phenylketonuria.

Maximum inhibition of phenylalanine hydroxylase activity in the liver (85%) and in the kidney (50%) of suckling rats required the administration of over 9 mumol of p-chlorophenylalanine/10g body weight. Despite the decrease in the total activity from 184 to 34 units per 10g body weight, the injection of as much as 26 mumol of phenylalanine was required for its concentration in plasma to be still considerably elevated 12h later. In rats injected with p-chlorophenylalanine every 48h and with phenylalanine every 24h from 3 to 18 days of age, the hepatic and renal phenylalanine hydroxylase remained inhibited, whereas the activities of three other hepatic enzymes were unchanged. There was about 20% inhibition of brain and body growth, but no interference with the developmental formation of several cerebral enzymes (four dehydrogenases, hexokinase and glutaminase) was detected. In the course of this prolonged treatment, the phenylalanine concentrations in plasma increased gradually; on day 2 and day 8 (measured 12h after the last injection) they were 800 and 1395 nmol/ml respectively; on day 15, 12 and 18h after the usual injection, the values were 2030 and 1030 respectively as opposed to the 96 nmol in untreated rats. This degree of hyperphenylalaninaemia, persisting for 18h per day throughout a critical period of development, fulfils the primary criterion of a suitable animal model for phenylketonuria.     

Comparison of alpha-methylphenylalanine and p-chlorophenylalanine as inducers of chronic hyperphenylalaninaemia in developing rats.

alpha-Methylphenylalanine is a very weak competitive inhibitor of rat liver phenylalanine hydroxylase in vitro but a potent suppressor in vivo. The loss of the hepatic activity (the renal one is unaffected) becomes maximal (70-75% decrease; cf. control) 18h after the administration (per 10g body wt.) of 24 mumol of alpha-methylphenylalanine with or without 52 mumol of phenylalanine. Chronic suppression of hepatic phenylalanine hydroxylase was obtained by injections of alpha-methylphenylalanine plus phenylalanine to suckling rats, and by their addition to the diet after weaning. A series of comparisons of the effects of this treatment, and one with p-chlorophenylalanine, was then carried out. In both cases there was a rise (1.3-2-fold) in phenylalanine-pyruvate amino-transferase activity (but no change in four other enzyme activities) in the liver; in brain there was a rise in phosphoserine phosphatase activity, but the total activity and subcellular distribution of nine enzymes revealed no other abnormalities in cerebral development. Striking increases in the concentration of plasma phenylalanine during 26 of the 31 experimental days (with a transient fall at 18-22 days) were maintained by treatment with both analogues plus phenylalanine. However, p-chlorophenylalanine-treated animals had a 30-60% mortality rate and 27-52% decrease in body weight. Developing rats treated with alpha-methylphenylalanine, showing no growth deficit or signs of toxicity (e.g. cataracts), appear to be a more suitable model for the human disease of phenylketonuria. Their phenylalanine concentrations exhibited at least 20-40-fold increase during 50% of each of the first 18 days of life, and 30-fold after weaning.     

Effects of Folic Acid on the Phenylalanine Tolerance Test in Phenylketonuria

Phenylalanine tolerance tests were performed on four untreated children with phenylketonuria, aged 8 to 12 years, before and after 14 days of folic acid administration (70 mg./day). No significant changes were noted in the phenylalanine tolerance tests or in the general condition of the patients. This study was carried out because of recent findings that a folic acid derivative is a co-factor in the conversion of phenylalanine to tyrosine.     

Metabolism of aromatic acids by Polyporus hispidus

When grown on glucose as principal carbon source the culture medium of Polyporus hispidus was found to contain phenolic acids, including p-coumaric and caffeic acids. ¹⁴C-Studies indicated that phenylalanine is converted to cinnamic acid as well as to phenylpyruvic acid and tyrosine in cultures. Cell-free preparations of mycelium contained phenylalanine and tyrosine ammonia-lyse activities and were capable of effecting the hydroxylation of cinnamic, p-coumaric and benzoic acids.     

Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg²⁺ were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm³), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm³).Abbreviations PAL L-phenylalanine ammonia-lyase - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - ATPase ATP phosphohydrolase     

Recent breakthroughs in the study of salicylic acid biosynthesis

Salicylic acid is an important regulator of induced plant resistance to pathogens. Consequently, the biosynthesis of salicylic acid and its regulation has received a lot of attention. Salicylic acid can be made from phenylalanine via cinnamic and benzoic acid. Recently, genetic studies in Arabidopsis have shown that salicylic acid is made in the chloroplast from isochorismate, a pathway that is known to operate in prokaryotes.     

Failure of 3-hydroxy-3-phenylpropanoic acid and cinnamic acid to serve as precursors of tropic acid in Datura innoxia

Datura innoxia plants were fed the R- and S-isomers of [3-¹⁴C]-3-hydroxy-3-phenylpropanoic acid, and [3-¹⁴C]cinnamic acid along with dl-[4-³H]phenylalanine. The hyoscyamine and scopolamine isolated from the plants 7 days later were labeled with tritium, but devoid of ¹⁴C, indicating that 3-hydroxy-3-phenylpropanoic acid and cinnamic acid are not intermediates between phenylalanine and tropic acid. The [³H] tropic acid obtained by hydrolysis of the hyoscyamine was degraded and shown to have essentially all its tritium located at the para position of its phenyl group, a result consistent with previous work.     

Heterologous production of flavanones in<Emphasis Type="Italic"> Escherichia coli</Emphasis>: potential for combinatorial biosynthesis of flavonoids in bacteria

Chalcones, the central precursor of flavonoids, are synthesized exclusively in plants from tyrosine and phenylalanine via the sequential reaction of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:coenzyme A ligase (4CL) and chalcone synthase (CHS). Chalcones are converted into the corresponding flavanones by the action of chalcone isomerase (CHI), or non-enzymatically under alkaline conditions. PAL from the yeast Rhodotorula rubra, 4CL from an actinomycete Streptomyces coelicolor A3(2), and CHS from a licorice plant Glycyrrhiza echinata, assembled as artificial gene clusters in different organizations, were used for fermentation production of flavanones in Escherichia coli. Because the bacterial 4CL enzyme attaches CoA to both cinnamic acid and 4-coumaric acid, the designed biosynthetic pathway bypassed the C4H step. E. coli carrying one of the designed gene clusters produced about 450 μg naringenin/l from tyrosine and 750 μg pinocembrin/l from phenylalanine. The successful production of plant-specific flavanones in bacteria demonstrates the usefulness of combinatorial biosynthesis approaches not only for the production of various compounds of plant and animal origin but also for the construction of libraries of "unnatural" natural compounds. Dedicated to Professor Sir David Hopwood.     

Comparative metabolic activity related to flavonoid synthesis in leaves and flowers of Chrysanthemum morifolium in response to K deficiency

K limitation could decrease the flavonoids content in many Chinese traditional herbs. These results may be caused by the influence of K deficiency on the synthesis pathway of flavonoids. In this paper, we aim to study the influence of K deficiency on secondary metabolites and activities of phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4CL) and cinnamate 4-hydroxylase (C4H) enzymes in the process of flavonoid synthesis in Chrysanthemum morifolium Ramat. The results show that K deficiency decreased the flavonoid and chlorogenic acid contents slightly in flower of C. morifolium while the same effect was obtained on C4H and PAL. Total flavonoids content increased with the content of cinnamic acid and p-coumaric acid in the plant under K deficiency, respectively. The regression equations between content of flavonoids and cinnamic acid/ p-coumaric acid should be expressed in term of linear effects (R²?=?0.9809, P?<?0.001 for cinnamic acid and R²?=?0.9929, P?<?0.0001 for p-coumaric acid, respectively). But the effect of phenylalanine on flavonoids should be expressed in term of quadratic pattern (R²?=?0.9375, P?<?0.05). There were two kinds principal components from chorismate to coumaryl CoA synthesis process, principal 1 was the substrate (phenylalanine, cinnamic acid, p-coumaric acid and PAL) and principal 2 was the enzyme (4CL, C4H), the principal 1 was the domination principal in both K deficient (88.36% and 10.57%) and sufficient treatments (88.17% and 9.64%), however, the factor loading (correlation coefficient of measured targets and the corresponding principal component) in principal 1 was opposite under different K application.