Aminotransferase Gene Research Articles

Cloning, Expression and Purification of Yeast Aminotransferase

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway.α‐aminoadipate is made from α‐ ketoadipate via the action of a PLP‐dependent aminotransferase that has not been identified. Sequence alignments and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. Based upon the homology, the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferas and one putative aminotransferase of unkown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. The aromatic aminotransferase coded by the Aro 8 gene has Km values for L‐tyrosine and L‐phenylalanine of 0.5 and 1 mM respectively. The Kcat values are about 10 s‐1 for both substrates. The uv/vis spectra of the aromatic aminotransferases have been determined as a function of pH. Substrate titrations and initial rate studies have been done to determine the substrate specificity of these enzymes.

Abrogation of Glucocorticoid Receptor Dimerization Correlates with Dissociated Glucocorticoid Behavior of Compound A

Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid-responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [(3)H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization-deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GR(dim)) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.

Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.

Hepatocyte Nuclear Factor-4α Induces Transdifferentiation of Hematopoietic Cells into Hepatocytes

Hematopoietic stem cells can directly transdifferentiate into hepatocytes because of cellular plasticity, but the molecular basis of transdifferentiation is not known. Here, we show the molecular basis using lineage-depleted oncostatin M receptor beta-expressing (Lin(-)OSMRbeta(+)) mouse bone marrow cells in a hepatic differentiation culture system. Differentiation of the cells was marked by the expression of albumin. Hepatocyte nuclear factor (HNF)-4alpha was expressed and translocated into the nuclei of the differentiating cells. Suppression of its activation in OSM-neutralized culture medium inhibited cellular differentiation. Ectopic expression of full-length HNF4alpha in 32D myeloid cells resulted in decreased myeloid colony-forming potential and increased expression of hepatocyte-specific genes and proteins. Nevertheless, the neohepatocytes produced in culture expressed active P450 enzyme. The obligatory role of HNF4alpha in hepatic differentiation was confirmed by transfecting Lin(-)OSMRbeta(+) cells with dominant negative HNF4alpha in the differentiation culture because its expression inhibited the transcription of the albumin and tyrosine aminotransferase genes. The loss and gain of functional activities strongly suggested that HNF4alpha plays a central role in the transdifferentiation process. For the first time, this report demonstrates the mechanism of transdifferentiation of hematopoietic cells into hepatocytes, in which HNF4alpha serves as a molecular switch.

Identification and Partial Characterization of an L-Tyrosine Aminotransferase (TAT) fromArabidopsis thaliana

The aminotransferase gene family in the model plant Arabidopsis thaliana consists of 44 genes. Twenty six of these enzymes are classified as characterized meaning that the reaction(s) that the enzyme catalyzes are documented using experimental means. The remaining 18 enzymes are uncharacterized and are therefore deemed putative. Our laboratory is interested in elucidating the function(s) of the remaining putative aminotransferase enzymes. To this end, we have identified and partially characterized an aminotransferase (TAT) enzyme from Arabidopsis annotated by the locus tag At5g36160. The full-length cDNA was cloned and the purified recombinant enzyme was characterized using in vitro and in vivo experiments. In vitro analysis showed that the enzyme is capable of interconverting L-Tyrosine and 4-hydroxyphenylpyruvate, and L-Phenylalanine and phenylpyruvate. In vivo analysis by functional complementation showed that the gene was able to complement an E. coli with a background of aminotransferase mutations that confers auxotrophy for L-Tyrosine and L-Phenylalanine.

Newly Mutatedputative-aminotransferasein Nonpungent Pepper (Capsicum annuum) Results in Biosynthesis of Capsinoids, Capsaicinoid Analogues

Capsinoids make up a group of nonpungent capsaicinoid analogues produced in Capsicum fruits. They have bioactivities similar to those of capsaicinoids such as suppression of fat accumulation and antioxidant activity. Because of their low pungency, they are more palatable ingredients in dietary supplements than capsaicinoids. We recently reported that capsinoid biosynthesis is caused by nonsense mutation in a putative aminotransferase gene (p-AMT) in a nonpungent cultivar CH-19 Sweet. Here we report on the screening of nonpungent germplasm that revealed a nonpungent cultivar Himo, which contains high levels of capsinoids. We have shown that Himo has a recessive allele of p-amt, which contains a mutation different from that of CH-19 Sweet. Sequence analysis of p-amt in Himo revealed that a single-nucleotide substitution results in one amino acid substitution from cysteine to arginine in the pyridoxal 5-phosphate binding domain. Genetic analysis using a cleaved amplified polymorphic sequence marker confirmed that the p-AMT genotype was precisely cosegregated with capsinoid biosynthesis and nonpungency. Himo will provide a new natural source of capsinoids.

Polymorphisms within the 4-aminobutyrate aminotransferase gene are associated with long-latency somatosensory potentials in families vulnerable for affective disorders

Depression is known to frequently co-occur with somatic symptoms and data from the Munich Vulnerability Study pointed to somatization as a possible vulnerability marker for affective disorders observable before disease onset. In this context previous studies furthermore revealed long-latency somatosensory evoked potentials (SSEP) to be a potential marker for susceptibility to depressive disorder. Based on this evidence we performed a small family-based association study to investigate genetic associations between long-latency SSEP and polymorphisms within candidate gene regions of somatosensory pathways including the serotonergic, GABAergic as well as the substance P system. The sample was composed of families at high risk for affective disorders who were participating in the Munich Vulnerability Study. We observed significant associations between average SSEP response strength in the long-latency range and polymorphisms within the 3' untranslated region of the 4-aminobutyrate aminotransferase gene coding for a protein responsible for GABA degradation. These findings point to a possible role of GABA catabolism in processing of somatosensory stimuli as well as for bodily misperceptions in connection with vulnerability for affective disorders.

Primary Hyperoxaluria in Cats Is Caused by a Mutation in the Feline GRHPR Gene

Primary hyperoxaluria (PH) is a rare, inherited disease in humans resulting from mutations in the alanine:glyoxylate aminotransferase gene (PH1) or in the glyoxylate reductase (GRHPR) gene (PH2). A disease in cats, mimicking PH2, was described with an autosomal recessive mode of inheritance. Recently, we recognized lesions consistent with PH in kidneys from 3 kittens. Genomic DNA was extracted from 1 blood and 2 formalin-fixed kidney samples from the 3 affected kittens, from blood from the affected cats’ sire, and from blood from 2 healthy unrelated cats. The 9 feline GRHPR exons and intronic donor‐acceptor sites were amplified and sequenced. A point mutation G to A was identified at the acceptor site of intron 4. Affected cats were AA, normal cats GG, and the sire was heterozygous A/G. RNA from healthy, carrier, and affected cats was extracted and the GRHPR transcript sequenced revealing an exon 5 deletion in the affected transcript. The 89-bp deletion causes a frameshift and a premature stop codon 44 amino acids downstream, resulting in an anticipated 119 amino acids missing from the C-terminus of the affected cat protein. The unaffected cat expresses the normal transcript, whereas the carrier expressed both.

Cloning, Expression and Purification of Yeast Aminotransferase

Lysine is synthesized in Saccharomyces cerevisiae via the α‐aminoadipate pathway. α‐aminoadipate is made from α‐ketoadipate via the action of PLP‐dependent aminotransferase that has not been identified. Sequence alignment and a homology search for conserved amino acid residues were used to identify six aminotransferase genes in S. cerevisiae. On the basis of sequence homology the identified genes code for two aromatic aminotransferases, two aspartate aminotransferases, one kynurenine aminotransferase and one putative amiontransferase of unknown function. The cloned genes were obtained from the Harvard Institute of Proteomics Plasmid Repository. The genes were subcloned into expression vector pET16b. The expressed his‐tagged proteins have been purified using a Ni‐NTA affinity column. Kinetic characterization of these enzymes is at the early stages.

Over-expression of aspartate aminotransferase genes in rice resulted in altered nitrogen metabolism and increased amino acid content in seeds

Aspartate aminotransferase (AAT) is a key enzyme in the synthesis of amino acids. It plays an important role in regulating carbon and nitrogen metabolism in almost all organisms. In this study, we over-expressed in rice separately all three AAT genes from rice (OsAAT1~3) and one AAT gene from Escherichia coli (EcAAT). Over-expression was driven by the CaMV 35S promoter and constructs were introduced into rice by Agrobacterium tumefaciens-mediated transformation. Compared with control plants, the transformants showed significantly increased leaf AAT activity and greater seed amino acid and protein contents. No other phenotypic changes were observed. The total leaf AAT activities in plants over-expressing OsAAT1, OsAAT2, and EcAAT were 26.6, 23.6, and 19.6 A min(-1) mg(-1) FW (A: units of activity, defined as increase of absorbency per min per mg; FW: fresh weight), which were significantly higher than that in the wild-type control (17.7 A min(-1) mg(-1) FW). The amino acid content in seeds of transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT was 119.36, 115.36, and 113.72 mg g(-1), respectively, which were 16.1, 12.0, and 5.4% higher, respectively, than that in the control plants. The transgenic plants over-expressing OsAAT1, OsAAT2, and EcAAT had significantly higher protein contents (increased 22.2, 21.1, and 11.1%, respectively) than wild-type plants. No significant changes were found in leaf AAT activity, seed amino acid content or protein content in OsAAT3 over-expressed plants. The expression patterns of the three OsAAT genes and their different functions are also discussed.

Isolation and Identification of Differentially Expressed Genes in Sugarcane Infected by Ustilago scitaminea

The objective of this study was to survey the molecular mechanism of resistance to sugarcane smut ( Ustilago scitaminea Syd.). Two sugarcane ( Saccharum officinarum complex) varieties NCo376 with high resistance and F134 with susceptibility were infected by U. scitaminea and the genes associated with the smut resistance were detected with 12 anchored primers and 8 random primers via differential display reverse transcription PCR (DDRT-PCR). Seven differentially expressed fragments were obtained through cloning, sequencing, and semiquantitative RT-PCR validation. The results of Blast in GenBank showed that they shared high homology (35–99%) with cytochrome C oxidase ( CCO) gene, ribosomal protein gene, NAD-dependent malic enzyme gene, aminotransferase gene, binding protein gene, RNA polymerase specific transcription initation factor and retrotransposon. The results of semiquantitative RT-PCR showed that CCO gene expression was regulated by U. scitaminea and salicylic acid, and was independent of H 2O 2. Besides, CCO gene was expressed in root, stalk, and leaf of sugarcane at relatively low levels. Thereby, the phytoalexin induced by CCO gene was inferred to inhibit the pathogen after infection.

Functional characterization of kanB by complementing in engineered Streptomyces fradiae ∆neo6::tsr

A putative aminotransferase gene, kanB, lies in the biosynthetic gene cluster of Streptomyces kanamyceticus ATCC 12853 and has 66% identity with neo6 in neomycin biosynthesis. Streptomyces fradiae Deltaneo6::tsr was generated by disrupting neo6 in the neomycin producer Streptomyces fradiae. Neomycin production was completely abolished in the disruptant mutant but was restored through self-complementation of neo6. S. fradiae HN4 was generated through complementation with kanB in Streptomyces fradiae neo6::tsr. Based on metabolite analysis by ESI/MS and LC/MS, neomycin production was restored in Streptomyces fradiae HN4. Thus, like neo6, kanB also functions as a 2-deoxy-scyllo-inosose aminotransferase that has dual functions in the formation of 2-deoxy-scyllo-inosose (DOS).

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Molecular characterization of mungbean peroxisomal alanine glyoxylate aminotransferase gene induced by low temperature stress

Photorespiration reduces carbon fixation rate, but it is an essential process in plant. Photorespiration involves reactions in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes the conversion of alanine and glyoxylate into glycine and pyruvate, respectively. We isolated a low temperature-inducible cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated asMLT92, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed that the mungbean genome has one copy ofMLT92. MLT92 mRNA was induced by low temperature and its RNA expression was not much increased by drought stress. ABA and NaCl did not induce RNA expression ofMLT92. Based on GFP/RFP targeting experiment, GFP-MLT92 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome in tobacco protoplasts. This suggests that peroxisomalMLT92 is involved in molecular response to low temperature stress.

Effects of fenofibrate on plasma and hepatic transaminase activities and hepatic transaminase gene expression in rats

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are widely used as sensitive markers of possible tissue damage, particularly liver toxicity. Lipid-lowering drugs, such as fibrates, slightly increase serum transaminase levels in humans, but there is little evidence that the phenomenon is related to drug-induced liver injury (DILI). Some in vitro studies have indicated that the elevations of serum transaminase activities after treatment of humans with fenofibrate, one of the fibrates, are related to increased transaminase synthesis in the hepatocytes rather than to transaminase leakage from the hepatocytes associated with cell lysis. In this study, male F344/DuCrlCrlj (Fischer) rats were treated once with fenofibrate at a dose level of 400 mg/kg and the relationships between the pharmacological effects, blood and hepatic transaminase activities and the gene expression of the transaminases in the liver were investigated. Fenofibrate treatment slightly increased plasma transaminase activities in rats with the findings directly related to the pharmacological action of the drug. The increases were in parallel with increases in hepatic transaminase activities associated with increases in the transaminase genes in the liver and were not considered to be a consequence of hepatotoxicity from the drug. The modification in transaminase gene expression is likely to be secondary to the pharmacological action of fenofibrate. The evidence obtained in our study underlines the importance of gene regulation as a possible alternative mechanism for increased blood transaminase activities.

Lactate and Amino Acid Catabolism in the Cheese-Ripening Yeast Yarrowia lipolytica

The consumption of lactate and amino acids is very important for microbial development and/or aroma production during cheese ripening. A strain of Yarrowia lipolytica isolated from cheese was grown in a liquid medium containing lactate in the presence of a low (0.1x) or high (2x) concentration of amino acids. Our results show that there was a dramatic increase in the growth of Y. lipolytica in the medium containing a high amino acid concentration, but there was limited lactate consumption. Conversely, lactate was efficiently consumed in the medium containing a low concentration of amino acids after amino acid depletion was complete. These data suggest that the amino acids are used by Y. lipolytica as a main energy source, whereas lactate is consumed following amino acid depletion. Amino acid degradation was accompanied by ammonia production corresponding to a dramatic increase in the pH. The effect of adding amino acids to a Y. lipolytica culture grown on lactate was also investigated. Real-time quantitative PCR analyses were performed with specific primers for five genes involved in amino acid transport and catabolism, including an amino acid transporter gene (GAP1) and four aminotransferase genes (ARO8, ARO9, BAT1, and BAT2). The expression of three genes involved in lactate transport and catabolism was also studied. These genes included a lactate transporter gene (JEN1) and two lactate dehydrogenase genes (CYB2-1 and CYB2-2). Our data showed that GAP1, BAT2, BAT1, and ARO8 were maximally expressed after 15 to 30 min following addition of amino acids (BAT2 was the most highly expressed gene), while the maximum expression of JEN1, CYB2-1, and CYB2-2 was delayed (>or=60 min).

Characterization of the 3-HKT gene in important malaria vectors in India, viz: Anopheles culicifacies and Anopheles stephensi (Diptera: Culicidae)

The 3-hydroxykynurenine transaminase (3-HKT) gene plays a vital role in the development of malaria parasites by participating in the synthesis of xanthurenic acid, which is involved in the exflagellation of microgametocytes in the midgut of malaria vector species. The 3-HKT enzyme is involved in the tryptophan metabolism of Anophelines. The gene had been studied in the important global malaria vector, Anopheles gambiae. In this report, we have conducted a preliminary investigation to characterize this gene in the two important vector species of malaria in India, Anopheles culicifacies and Anopheles stephensi. The analysis of the genetic structure of this gene in these species revealed high homology with the An. gambiae gene. However, four non-synonymous mutations in An. stephensi and seven in An. culicifacies sequences were noted in the exons 1 and 2 of the gene; the implication of these mutations on enzyme structure remains to be explored.

Identification and Diversity of Putative Aminoglycoside-Biosynthetic Aminotransferase Genes from Deep-Sea Environmental DNA

We obtained DNA fragments encoding putative aminotransferases possibly involved in the biosynthesis of aminoglycoside antibiotics from deep-sea sediments of the northwest Pacific Ocean by nested PCR, and 34 individual genes (total 89 clones) were identified. About half of the deep-sea sequences showed similarity with genes of known aminoglycoside-producers, but others were deep-sea specific genes. Furthermore, we found that temperature-gradient gel electrophoresis (TGGE) can be an effective tool in the analysis of these DNA fragments.

A Novel NAC Transcription Factor, IDEF2, That Recognizes the Iron Deficiency-responsive Element 2 Regulates the Genes Involved in Iron Homeostasis in Plants

Iron is essential for most living organisms, and thus iron deficiency poses a major abiotic stress in crop production. Plants induce iron utilization systems under conditions of low iron availability, but the molecular mechanisms of gene regulation under iron deficiency remain largely unknown. We identified a novel transcription factor of rice and barley, IDEF2, which specifically binds to the iron deficiency-responsive cis-acting element 2 (IDE2) by yeast one-hybrid screening. IDEF2 belongs to an uncharacterized branch of the NAC transcription factor family and exhibits novel properties of sequence recognition. An electrophoretic mobility shift assay and cyclic amplification and selection of targets experiment revealed that IDEF2 predominantly recognized CA(A/C)G(T/C)(T/C/A)(T/C/A) within IDE2 as the core-binding site. IDEF2 transcripts are constitutively present in rice roots and leaves. Repression of the function of IDEF2 by the RNA interference (RNAi) technique and chimeric repressor gene-silencing technology (CRES-T) caused aberrant iron homeostasis in rice. Several genes up-regulated by iron deficiency, including the Fe(II)-nicotianamine transporter gene OsYSL2, were less induced by iron deficiency in the RNAi rice of IDEF2, suggesting that IDEF2 is involved in the regulation of these genes. Many genes with repressed expression in IDEF2 RNAi rice possessed the IDEF2-binding core sites in their promoters, and the flanking sequences were also highly homologous to IDE2. IDEF2 bound to OsYSL2 promoter region containing the binding core site, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

Richner–Hanhart Syndrome Detected by Expanded Newborn Screening

Richner-Hanhart syndrome (tyrosinemia type 2) is an inborn error of tyrosine metabolism which is clinically characterized mainly by oculocutaneous symptoms including corneal opacities and keratosis palmoplantaris. Skin symptoms usually develop after the first year of life. We report a neonate in whom already on the third day of life diagnosis of Richner-Hanhart syndrome could be suspected because of elevated tyrosine levels in newborn screening by tandem mass spectrometry. Analysis of the tyrosine aminotransferase gene revealed a homozygous missense mutation p.R433W (c.1297C>T). An 8-year-old brother with persistent plantar hyperkeratotic plaques of the soles of yet unknown origin was subsequently identified to be also affected with Richner-Hanhart syndrome. This demonstrates that early diagnosis of Richner-Hanhart syndrome is possible in neonates by extended newborn screening. Early introduction of dietary treatment is a prerequisite to reduce the risk of clinical symptoms.