Identification and characterization of a novel tyrosine aminotransferase gene (SmTAT3-2) promotes the biosynthesis of phenolic acids in Salvia miltiorrhiza Bunge.

Rosmarinic acid (RA) and salvianolic acid B (SAB) are main phenolic acids in Salvia miltiorrhiza Bunge have been widely used in the treatment of cardiovascular and cerebrovascular diseases due to their excellent pharmacological activity. RA is a precursor of SAB, and tyrosine transaminase (TAT, EC 2.6.1.5) is a crucial rate-limiting enzyme in their metabolism pathway. This study identified a novel TAT gene, SmTAT3-2, and found that it is a new transcript derived from unconventional splicing of SmTAT3. We used different substrates for enzymatic reaction with SmTAT1, SmTAT3 and SmTAT3-2. Subcellular localization of SmTAT1 and SmTAT3-2 was completed based on submicroscopic techniques. In addition, they were overexpressed and CRISPR/Cas9 gene edited in hairy roots of S. miltiorrhiza. Revealed SmTAT3-2 and SmTAT1 showed a stronger affinity for L-tyrosine than SmTAT3, localized in the cytoplasm, and promoted the synthesis of phenolic acid. In overexpressed SmTAT3-2 hairy roots, the content of RA and SAB was significantly increased by 2.53 and 3.38 fold, respectively, which was significantly higher than that of overexpressed SmTAT1 strain compared with EV strain. These findings provide a valuable key enzyme gene for the phenolic acids metabolism pathway and offer a theoretical basis for the clinical application.

Expression Patterns and Functional Analysis of Three SmTAT Genes Encoding Tyrosine Aminotransferases in Salvia miltiorrhiza.

Tyrosine aminotransferase (TAT, E.C. 2.6.1.5) is a pyridoxal phosphate-dependent aminotransferase that is widely found in living organisms. It catalyzes the transfer of the amino group on tyrosine to α-ketoglutarate to produce 4-hydroxyphenylpyruvic acid (4-HPP) and is the first enzyme for tyrosine degradation. Three SmTATs have been identified in the genome of Salvia miltiorrhiza (a model medicinal plant), but their information is very limited. Here, the expression profiles of the three SmTAT genes (SmTAT1, SmTAT2, and SmTAT3) were studied. All three genes expressed in different tissues and responded to methyl jasmonate stimuli. SmTAT proteins are localized in the cytoplasm. The recombinant SmTATs were subjected to in vitro biochemical properties. All three recombinant enzymes had TAT activities and SmTAT1 had the highest catalytic activity for tyrosine, followed by SmTAT3. Also, SmTAT1 preferred the direction of tyrosine deamination to 4-HPP, while SmTAT2 preferred transamination of 4-HPP to tyrosine. In parallel, transient overexpression of SmTATs in tobacco leaves revealed that all three SmTAT proteins catalyzed tyrosine to 4-HPP in vivo, with SmTAT1 exhibiting the highest enzymatic activity. Overall, our results lay a foundation for the production of tyrosine-derived secondary metabolites via metabolic engineering or synthetic biology in the future.

Molecular cloning and characterization of tyrosine aminotransferase and hydroxyphenylpyruvate reductase, and rosmarinic acid accumulation in Scutellaria baicalensis.

Rosmarinic acid (a-O-caffeoyl-3,4-dihydroxyphenylacetic acid, RA) is a caffeoyl ester widely distributed in plants. cDNA clones encoding tyrosine aminotransferase (TAT1 and 2) and hydroxyphenylpyruvate reductase (HPPR) have been isolated from Scutellaria baicalensis. The open reading frames (ORFs) of SbTAT1 and 2 were 1230 and 1272 bp long and encoded 409 and 423 amino acid residues, respectively. HPPR corresponded to a 942-bp ORF and 313 amino acid residues of translated protein. To study the molecular mechanisms of TAT and HPPR and investigate RA accumulation in S. baicalensis, we examined the transcript levels of TAT isoforms and HPPR with quantitative real-time PCR and analyzed the RA content in different organs by using high-performance liquid chromatography. The transcript levels of SbTATI SbTAT2, and SbHPPR in the flowers were higher than those in other organs. RA was also highly accumulated in the flowers and with a trace amount in the roots. No RA was detected in the leaves and stems of S. baicalensis. The amount of accumulated RA in the flowers was 28.7 times higher than that in the roots. Our results will be helpful in elucidating the mechanisms of RA biosynthesis in S. baicalensis.

TATN-1 mutations reveal a novel role for tyrosine as a metabolic signal that influences developmental decisions and longevity in Caenorhabditis elegans.

Recent work has identified changes in the metabolism of the aromatic amino acid tyrosine as a risk factor for diabetes and a contributor to the development of liver cancer. While these findings could suggest a role for tyrosine as a direct regulator of the behavior of cells and tissues, evidence for this model is currently lacking. Through the use of RNAi and genetic mutants, we identify tatn-1, which is the worm ortholog of tyrosine aminotransferase and catalyzes the first step of the conserved tyrosine degradation pathway, as a novel regulator of the dauer decision and modulator of the daf-2 insulin/IGF-1-like (IGFR) signaling pathway in Caenorhabditis elegans. Mutations affecting tatn-1 elevate tyrosine levels in the animal, and enhance the effects of mutations in genes that lie within the daf-2/insulin signaling pathway or are otherwise upstream of daf-16/FOXO on both dauer formation and worm longevity. These effects are mediated by elevated tyrosine levels as supplemental dietary tyrosine mimics the phenotypes produced by a tatn-1 mutation, and the effects still occur when the enzymes needed to convert tyrosine into catecholamine neurotransmitters are missing. The effects on dauer formation and lifespan require the aak-2/AMPK gene, and tatn-1 mutations increase phospho-AAK-2 levels. In contrast, the daf-16/FOXO transcription factor is only partially required for the effects on dauer formation and not required for increased longevity. We also find that the controlled metabolism of tyrosine by tatn-1 may function normally in dauer formation because the expression of the TATN-1 protein is regulated both by daf-2/IGFR signaling and also by the same dietary and environmental cues which influence dauer formation. Our findings point to a novel role for tyrosine as a developmental regulator and modulator of longevity, and support a model where elevated tyrosine levels play a causal role in the development of diabetes and cancer in people.

Tyrosine aminotransferase contributes to benzylisoquinoline alkaloid biosynthesis in opium poppy.

Tyrosine aminotransferase (TyrAT) catalyzes the transamination of L-Tyr and α-ketoglutarate, yielding 4-hydroxyphenylpyruvic acid and L-glutamate. The decarboxylation product of 4-hydroxyphenylpyruvic acid, 4-hydroxyphenylacetaldehyde, is a precursor to a large and diverse group of natural products known collectively as benzylisoquinoline alkaloids (BIAs). We have isolated and characterized a TyrAT cDNA from opium poppy (Papaver somniferum), which remains the only commercial source for several pharmaceutical BIAs, including codeine, morphine, and noscapine. TyrAT belongs to group I pyridoxal 5'-phosphate (PLP)-dependent enzymes wherein Schiff base formation occurs between PLP and a specific Lys residue. The amino acid sequence of TyrAT showed considerable homology to other putative plant TyrATs, although few of these have been functionally characterized. Purified, recombinant TyrAT displayed a molecular mass of approximately 46 kD and a substrate preference for L-Tyr and α-ketoglutarate, with apparent K(m) values of 1.82 and 0.35 mm, respectively. No specific requirement for PLP was detected in vitro. Liquid chromatography-tandem mass spectrometry confirmed the conversion of L-Tyr to 4-hydroxyphenylpyruvate. TyrAT gene transcripts were most abundant in roots and stems of mature opium poppy plants. Virus-induced gene silencing was used to evaluate the contribution of TyrAT to BIA metabolism in opium poppy. TyrAT transcript levels were reduced by at least 80% in silenced plants compared with controls and showed a moderate reduction in total alkaloid content. The modest correlation between transcript levels and BIA accumulation in opium poppy supports a role for TyrAT in the generation of alkaloid precursors, but it also suggests the occurrence of other sources for 4-hydroxyphenylacetaldehyde.

Characterization and expression profiling of tyrosine aminotransferase gene from Salvia miltiorrhiza (Dan-shen) in rosmarinic acid biosynthesis pathway.

A novel tyrosine aminotransferase gene (designated as SmTAT) involved in rosmarinic acid biosynthesis pathway is cloned from Salvia miltiorrhiza Bung. The full-length cDNA of SmTAT is 1,603 bp long with an open reading frame (ORF) of 1,233 bp encoding a polypeptide of 411 amino acid residues. The deduced amino acid sequence of the SmTAT gene shared high homology with other known TATs. Analysis of SmTAT genomic DNA reveals that it contains 6 exons, 5 introns. The analysis of SmTAT promoter region and terminator region was also presented. Semi-quantitative RT-PCR analysis reveals that the constitutive expression of SmTAT in stem is much higher than that in root, leaf. Further expression analysis reveals that the signaling components of defense/stress pathways, such as methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA) and ultraviolet-B radiation (UV-B), up-regulate the SmTAT transcript levels over the control. This study provides useful information for further studying this gene and its function in rosmarinic acid biosynthetic pathway in S. miltiorrhiza, the roots of which so-called ''Danshen'' possess many pharmaceutical properties for human health.

Isolation, culture and immortalisation of hepatic oval cells from adult mice fed a choline-deficient, ethionine-supplemented diet.

Oval cells have great potential for use in cell therapy to treat liver disease, however this cannot be achieved until the factors which govern their proliferation and differentiation are better understood. We describe a method to establish primary cultures of murine oval cells, and the derivation of two novel lines from these. Primary cultures from the livers of wildtype or TAT-GRE lacZ transgenic mice subjected to a choline-deficient, ethionine-supplemented diet comprised up to 80% oval cells at day 7 based on A6 or CK19 staining. Cell lines were clonally derived, which underwent spontaneous immortalisation following prolonged maintenance in culture. Immunostaining and RT-PCR demonstrated they express hepatocytic and biliary markers and they were therefore termed "bipotential murine oval liver" (BMOL) cells. Under proliferating culture conditions, BMOL or BMOL-TAT cells abundantly expressed oval cell and biliary markers, whereas mature hepatocytic markers were upregulated when the growth conditions were changed to facilitate differentiation. Hepatic differentiation of BMOL-TAT cells could be traced by measuring the expression of their lacZ transgene, which is driven by a promoter element from tyrosine aminotransferase (TAT), a marker of adult hepatocytes. Interestingly, haematopoietic markers were upregulated in superconfluent cultures, indicating a possible multipotentiality. None of the cell lines grew in semi-solid agar, nor did they form tumours in nude mice, suggesting they are non-tumourigenic. These novel murine oval cell lines, together with a reliable method for isolation and culture of primary oval cells, will provide a useful tool for investigating the contribution of oval cells to liver regeneration.

Comparison of the tyrosine aminotransferase cDNA and genomic DNA sequences of normal mink and mink affected with tyrosinemia type II.

Type II tyrosinemia, designated Richner-Hanhart syndrome in humans, is a hereditary metabolic disorder with autosomal recessive inheritance characterized by a deficiency of tyrosine aminotransferase activity. Mutations occur in the human tyrosine aminotransferase gene, resulting in high levels of tyrosine and disease. Type II tyrosinemia occurs in mink, and our hypothesis was that it would also be associated with mutation(s) in the tyrosine aminotransferase gene. Therefore, the transcribed cDNA and the genomic tyrosine aminotransferase gene were sequenced from normal and affected mink. The gene extended over 11.9 kb and had 12 exons coding for a predicted 454-amino-acid protein with 93% homology with human tyrosine aminotransferase. FISH analysis mapped the gene to chromosome 8 using the Mandahl and Fredga (1975) nomenclature and chromosome 5 using the Christensen et al. (1996) nomenclature. The hypothesis was rejected because sequence analysis disclosed no mutations in either cDNA or introns that were associated with affected mink. This suggests that an unlinked gene regulatory mutation may be the cause of tyrosinemia in mink.

Cloning and characterization of a coronatine-regulated tyrosine aminotransferase from Arabidopsis.

In plants, the phytotoxin coronatine, which is an analog of the octadecanoids 12-oxo-phytodienoic acid and/or jasmonic acid, gives rise to a number of physiological responses similar to those of octadecanoids. To further elucidate the physiological role of these compounds, the differential RNA display technique was used to isolate a number of novel octadecanoid-inducible genes expressed in coronatine-treated Arabidopsis. Among these, a cDNA clone was identified that was similar to known tyrosine aminotransferases (TATs). The function was verified with the expressed recombinant protein. In Arabidopsis, the protein is present as a multimer of 98 kD, with a monomer of an apparent molecular mass of 47 kD. TAT mRNA could be induced within 2 h by various octadecanoids and by wounding of the plants. Accumulation of the TAT protein and a 5- to 7-fold increase in its enzymatic activity was observed 7 to 9 h after application of octadecanoids, coronatine, or wounding. The potential role of TAT in the defense response to herbivores and pathogens is discussed.

Hyperthermic stress affects glucocorticoid receptor-mediated transcription in rat liver.

Binding capacity of the cytoplasmic and nuclear glucocorticoid receptor (GR) and the activity of tyrosine aminotransferase (TAT) were examined in the liver of intact and adrenalectomized rats exposed to 41 degrees C whole body hyperthermic stress. In glucocorticoid-deprived animals, stress-induced decrease in the cytoplasmic steroid binding was followed by parallel increases in its nuclear binding and TAT activity, suggesting a stimulation of TAT gene transcription by the GR in the absence of the ligand. In intact animals, however, a diminution of the steroid binding in the cytosol, its unchanged nuclear binding and an impairment of TAT activity were observed upon the stress. The results imply that stress could elicit different structural or functional alterations of unliganded vs liganded GR.

Cloning and expression of human tyrosine aminotransferase cDNA.

Complementary DNA clones encoding human tyrosine aminotransferase (TAT) were isolated by screening a normal adult woman liver lambda gt11 library with rat TAT cDNA. The largest isolated cDNA is 2051 bp long (EMBL accession number X55675). This cDNA was subcloned downstream of the cytomegalovirus promoter in the pCMV vector for transfection into human cervical carcinoma HeLa cells. Expression of the TAT cDNA resulted in the synthesis of a protein with a molecular mass of approximately 50 kDa, as assessed by Western analysis, a value which is in close agreement with the predicted molecular weight of 50,399, for a deduced sequence of 454 amino acids. The expressed protein catalyzed specifically the conversion of L-[14C]tyrosine into p-[14C]hydroxyphenylpyruvate. The availability of a functional TAT cDNA provides a useful tool for detailed study of the structure-function relationship of the enzyme and its mutated derivatives.

Ginsenoside-Rg1 regulates the induction of tyrosine aminotransferase gene transcription in rat hepatocyte cultures.

Ginsenosides present in the roots of Panax ginseng C.A. Meyer have been shown to induce a number of hepatocyte gene expression. We have recently demonstrated that ginsenoside-Rg1 (G-Rg1) stimulated the enzyme activity of tyrosine aminotransferase (TAT), a hepatocyte specific enzyme, of which enzyme activity was dose-dependently inhibited by RU486, a specific glucocorticoid antagonist. This study was therefore designed to determine whether G-Rg1 induces the transcriptional activity of TAT gene and to investigate whether G-Rg1 induces the gene transcription by glucocorticoid receptor- or cAMP-mediated induction mechanism. The slot blot hybridization analysis revealed that the TAT-mRNA level was increased by 9.3-fold in hepatocyte cultures in response to G-Rg1 stimulation. In contrast, the inductive effect of G-Rg1 was almost equally inhibited, that is, by 49% or 50% respectively in the presence of RU486 or Rp-cAMPs, a specific competitive inhibitor of protein kinase A. These results in hepatocyte cultures suggest that G-Rg1 modulates the TAT gene transcription through its influence on a functional or cooperative interaction between glucocorticoid receptor- and cAMP-mediated induction mechanism.

Insulin antagonism of dexamethasone induction of tyrosine aminotransferase in cultured fetal hepatocytes. A correlation between enzyme activity, synthesis, level of messenger RNA and transcription.

Previous studies have shown that insulin depresses the induction of tyrosine aminotransferase by glucocorticoids in cultured fetal rat hepatocytes. However, the site at which this inhibitory effect is exerted was not elucidated, since only enzyme activity was determined in such studies. Therefore, the effect of insulin on tyrosine aminotransferase synthesis, the level of its mRNA as well as the rate of transcription of the gene in isolated nuclei have been determined. The results obtained indicate that in cultures exposed to dexamethasone, Bt2cAMP, insulin and combinations of these additives, there is an excellent correlation between the enzyme activity, enzyme synthesis and the level of mRNA. Run-on transcription experiments indicate that the reduction in the level of mRNA by insulin in dexamethasone-supplemented cultures is the result of a diminished rate of gene transcription.

The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover.

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.

Complete complementary DNA of rat tyrosine aminotransferase messenger RNA. Deduction of the primary structure of the enzyme.

The primary structure of rat tyrosine aminotransferase (L-tyrosine:2-oxoglutarate aminotransferase; EC 2.6.1.5), a liver-specific enzyme involved in gluconeogenesis, has been deduced from the nucleotide sequence of a cloned full-length cDNA. The mRNA is 2362 nucleotides long (excluding the poly(A) tail) and codes for a polypeptide of 454 amino acids with a molecular weight of 50634. Unambiguous identification was obtained by comparison of this sequence with the amino acid sequences of several peptides obtained from the purified enzyme.