A Phenylpyruvic Acid Reductase Is Required for Biosynthesis of Tropane Alkaloids.

Solanaceous medicinal plants produce tropane alkaloids (TAs). We discovered a novel gene from Atropa belladonna, AbPPAR, which encodes a phenylpyruvic acid reductase required for TA biosynthesis. AbPPAR was specifically expressed in root pericycles and endodermis. AbPPAR was shown to catalyze reduction of phenylpyruvic acid to phenyllactic acid, a precursor of TAs. Suppression of AbPPAR disrupted TA biosynthesis through reduction of phenyllactic acid levels. In summary, we identified a novel enzyme involved in TA biosynthesis.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA3, MeJA and Ag+, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Phenylalanine sensitive K562-D cells for the analysis of the biochemical impact of excess amino acid.

Although it is recognized that the abnormal accumulation of amino acid is a cause of the symptoms in metabolic disease such as phenylketonuria (PKU), the relationship between disease severity and serum amino acid levels is not well understood due to the lack of experimental model. Here, we present a novel in vitro cellular model using K562-D cells that proliferate slowly in the presence of excessive amount of phenylalanine within the clinically observed range, but not phenylpyruvate. The increased expression of the L-type amino acid transporter (LAT2) and its adapter protein 4F2 heavy chain appeared to be responsible for the higher sensitivity to phenylalanine in K562-D cells. Supplementation with valine over phenylalanine effectively restored cell proliferation, although other amino acids did not improve K562-D cell proliferation over phenylalanine. Biochemical analysis revealed mammalian target of rapamycin complex (mTORC) as a terminal target of phenylalanine in K562-D cell proliferation, and supplementation of valine restored mTORC1 activity. Our results show that K562-D cell can be a potent tool for the investigation of PKU at the molecular level and to explore new therapeutic approaches to the disease.

Resolving phenylalanine metabolism sheds light on natural synthesis of penicillin G in Penicillium chrysogenum.

The industrial production of penicillin G by Penicillium chrysogenum requires the supplementation of the growth medium with the side chain precursor phenylacetate. The growth of P. chrysogenum with phenylalanine as the sole nitrogen source resulted in the extracellular production of phenylacetate and penicillin G. To analyze this natural pathway for penicillin G production, chemostat cultures were switched to [U-(13)C]phenylalanine as the nitrogen source. The quantification and modeling of the dynamics of labeled metabolites indicated that phenylalanine was (i) incorporated in nascent protein, (ii) transaminated to phenylpyruvate and further converted by oxidation or by decarboxylation, and (iii) hydroxylated to tyrosine and subsequently metabolized via the homogentisate pathway. The involvement of the homogentisate pathway was supported by the comparative transcriptome analysis of P. chrysogenum cultures grown with phenylalanine and with (NH(4))(2)SO(4) as the nitrogen source. This transcriptome analysis also enabled the identification of two putative 2-oxo acid decarboxylase genes (Pc13g9300 and Pc18g01490). cDNAs of both genes were cloned and expressed in the 2-oxo-acid-decarboxylase-free Saccharomyces cerevisiae strain CEN.PK711-7C (pdc1 pdc5 pdc6Δ aro10Δ thi3Δ). The introduction of Pc13g09300 restored the growth of this S. cerevisiae mutant on glucose and phenylalanine, thereby demonstrating that Pc13g09300 encodes a dual-substrate pyruvate and phenylpyruvate decarboxylase, which plays a key role in an Ehrlich-type pathway for the production of phenylacetate in P. chrysogenum. These results provide a basis for the metabolic engineering of P. chrysogenum for the production of the penicillin G side chain precursor phenylacetate.

Identification of genes in the phenylalanine metabolic pathway by ectopic expression of a MYB transcription factor in tomato fruit.

Altering expression of transcription factors can be an effective means to coordinately modulate entire metabolic pathways in plants. It can also provide useful information concerning the identities of genes that constitute metabolic networks. Here, we used ectopic expression of a MYB transcription factor, Petunia hybrida ODORANT1, to alter Phe and phenylpropanoid metabolism in tomato (Solanum lycopersicum) fruits. Despite the importance of Phe and phenylpropanoids to plant and human health, the pathway for Phe synthesis has not been unambiguously determined. Microarray analysis of ripening fruits from transgenic and control plants permitted identification of a suite of coregulated genes involved in synthesis and further metabolism of Phe. The pattern of coregulated gene expression facilitated discovery of the tomato gene encoding prephenate aminotransferase, which converts prephenate to arogenate. The expression and biochemical data establish an arogenate pathway for Phe synthesis in tomato fruits. Metabolic profiling and ¹³C flux analysis of ripe fruits further revealed large increases in the levels of a specific subset of phenylpropanoid compounds. However, while increased levels of these human nutrition-related phenylpropanoids may be desirable, there were no increases in levels of Phe-derived flavor volatiles.

Production of 4-hydroxyphenyllactic acid by Lactobacillus sp. SK007 fermentation.

The production of 4-hydroxyphenyllactic acid (4-HO-PLA), a novel antifungal compound, was studied in Lactobacillus sp. SK007 growth. When grown in MRS broth, the strain could produce 75 microg/ml HO-4-PLA, which was the highest reported so far. Tyrosine and 4-hydroxyphenylpyruvic acid (HO-4-PPA) supplements during fermentation could both increase the HO-4-PLA production yield, and the effect of HO-4-PPA on HO-4-PLA production was remarkably better than that of tyrosine. Using HO-4-PPA as substrate could effectively produce HO-4-PLA, which reached 1.26 mg/ml.

Plant-derived anti-inflammatory compounds affect MIF tautomerase activity.

The cytokine macrophage migration inhibitory factor (MIF) has recently emerged as a crucial factor in the pathogenesis of rheumatoid arthritis (RA). It is debated whether the MIF mediated tautomeric conversion of either phenylpyruvate or of its other phenolic substrates is implicated in the pro-inflammatory action of this cytokine. Traditional herbal remedies have been used for centuries to alleviate inflammatory ailments of many kinds including arthritis. Several of their active ingredients identified are mono- or poly-phenol derivatives. In the present study the effect of some anti-inflammatory plant phenols on MIF mediated tautomerism of phenylpyruvate was investigated. Curcumin and caffeic acid were found to be the most potent inhibitors, exhibiting IC(50) values in the submicromolar range in the ketonase assay. Resveratrol and umbelliferon were almost as potent inhibitors as the antipyretic-analgetic drug acetaminophen. Our results reveal MIF as a possible target for the herbal anti-rheumatic agents.

Effect of dietary threonine supplementation on tyrosine toxicity in the rat.

The objective of this study was to determine the effect of threonine supplementation on tyrosine metabolism in rats fed a low protein diet with excess tyrosine. The growth retardation and the development of eye and paw lesions that occur in rats ingesting a basal plus 3% or 5% L-tyrosine diet could be alleviated partially by the addition of 0.5% or 1.0% L-threonine to the diet. An increased blood tyrosine level in rats fed excess tyrosine was also lowered by threonine supplementation. In tyrotoxic conditions, the activities of liver tyrosine transaminase (EC 2.6.1.5) and threonine dehydratase (EC 4.2.9.16) were elevated, but p-hydroxyphenyl pyruvic acid oxidase (EC 1.13.11.27) which is also intimately associated with tyrosine toxicity was found to be inactivated. Furthermore, biosynthesis of ascorbic acid in liver was significantly lowered in this condition. However, addition of L-threonine in the diet, not only could cure the signs developed due to excess tyrosine, but also could affect the levels of enzymes studied.

Efficiency of phenylpyruvic and phenyllactic acids as substitutes for phenylalanine in the diet of the growing rat.

Male weanling albino rats fed a diet containing all nutrients for optimal growth, including 45.4 mumoles of phenylalanine/g, gained weight at an average of 6.5 g/day. Removal of phenylalanine caused an average weight loss of 1.1 g/day. Addition of graduated increments of phenylalanine led to progressive increases in growth rate; the increases being proportional to the dietary content of this amino acid. When phenylpyruvic or L-phenyllactic acids were added isonitrogenously to the phenyl-alanine-free diet, growth rate also increased. Percent efficiency of these amino acid analogues as dietary substitutes for phenylalanine, calculated as (see article) varied from 50% to 70% for phenylpyruvic acid and 65% to 95% for L-phenyllactic acid. Efficiency increased as dose of analogue increased. Non-isonitrogenous substitution did not change growth rates. Supplementation with a megadose of pyridoxine did not increase the efficiency of phenylpyruvic acid. D-phenyllactic acid and cinnamic acid were completely ineffective as substitutes for phenylalanine. When tyrosine was withdrawn from the diet, efficiency of phenylpyruvic acid was 70% at several doses. The efficiency of L-phenyllactic acid was 65 to 40%, decreasing with increasing dose.