Aminotransferase Gene Research Articles

OAT mutations and clinical features in two Japanese brothers with gyrate atrophy of the choroid and retina

Gyrate atrophy (GA) of the choroid and retina is an extremely rare inherited chorioretinal dystrophy. Ornithine aminotransferase (OAT) gene mutations are identified in patients with GA. The purpose of this study was to report a novel deletion mutation of the OAT gene and describe clinical features of two brothers with GA in a Japanese family. We performed ophthalmic examinations, including best-corrected visual acuity, slit-lamp biomicroscopy, dilated funduscopy, fundus autofluorescence imaging, optical coherence tomography, visual field testing, and full-field electroretinography (ERG). Serum ornithine concentrations and OAT activities were analyzed. Mutation screening of the OAT gene was performed using Sanger sequencing. Both brothers had compound heterozygous mutations (p.K169DfsX10 and p.R426X), one of which was novel. Their unaffected parents carried one of the mutations heterozygously. An arginine-restricted diet was started in the younger brother at the age of 2 years, while the diet was not initiated in the older brother until the age of 6 years. After more than 15 years of follow-up, the dietary treatment seemed to slow the progression of the chorioretinal lesions in the younger brother. However, when compared at the same age, the younger brother had more reduced ERG amplitudes and constricted visual fields than his older brother. We identified a novel frameshift mutation (p.K169DfsX10) in the OAT gene. While an early arginine-restricted dietary treatment suppressed the fundus changes of GA to some degree in the younger brother, the efficacy of suppressing the progression of visual function loss could not be clearly determined.

α-1,3-N-acetylgalactose aminotransferase gene 539G>C mutation leads to the A2B isoform.

In this study, the phenotypic identification and molecular mechanism of one case of an A2B subtype pedigree was investigated. ABO blood groupings were identified by serological methods and sequence amplification was performed by polymerase chain reaction (PCR) using TA cloning and DNA sequencing analysis to identify the pedigree and the ABO gene haploid of the proband. There were both A and B antigens on the proband's red blood cells, and anti-A1 antibodies were found in the serum. Direct sequencing of the 6th and 7th exons of the ABO gene showed the A208/B101 genotype, and haploid determination revealed the A208 and B101 alleles. Compared with the A102 allele sequence, the A208 allele was mutated at the 539 G>C site. Pedigree analysis showed that the ABO blood phenotypes of the proband's father, mother, husband, and daughter were A2, B, AB, and A2B, respectively, and their genotypes were A208/O02, B101/B101, A102/B101, and A208/B101, respectively. The father of the proband had anti-A1 antibodies and the A208 allele of the proband was inherited from her father, which can be passed on to her daughter. The α-1, 3-N-acetylgalactose aminotransferase gene 539G>C mutation resulted in A2B phenotype generation, and individual serum contained the anti-A1 antibody.

L-Tyrosine Induces DNA Damage in Brain and Blood of Rats

Mutations in the tyrosine aminotransferase gene have been identified to cause tyrosinemia type II which is inherited in an autosomal recessive manner. Studies have demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. Considering that the L-tyrosine may promote oxidative stress, the main objective of this study was to investigate the in vivo effects of L-tyrosine on DNA damage determined by the alkaline comet assay, in brain and blood of rats. In our acute protocol, Wistar rats (30 days old) were killed 1 h after a single intraperitoneal L-tyrosine injection (500 mg/kg) or saline. For chronic administration, the animals received two subcutaneous injections of L-tyrosine (500 mg/kg, 12-h intervals) or saline administered for 24 days starting at postnatal day (PD) 7 (last injection at PD 31), 12 h after the last injection, the animals were killed by decapitation. We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood when compared to control group. Moreover, we observed that chronic administration of L-tyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood when compared to control group. In conclusion, the present work demonstrated that DNA damage can be encountered in brain from animal models of hypertyrosinemia, DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder and to reinforce the role of oxidative stress in the pathophysiology of tyrosinemia type II.

Deciphering the Role of Aspartate and Prephenate Aminotransferase Activities in Plastid Nitrogen Metabolism

Chloroplasts and plastids of nonphotosynthetic plant cells contain two aspartate (Asp) aminotransferases: a eukaryotic type (Asp5) and a prokaryotic-type bifunctional enzyme displaying Asp and prephenate aminotransferase activities (PAT). We have identified the entire Asp aminotransferase gene family in Nicotiana benthamiana and isolated and cloned the genes encoding the isoenzymes with plastidic localization: NbAsp5 and NbPAT. Using a virus-induced gene silencing approach, we obtained N. benthamiana plants silenced for NbAsp5 and/or NbPAT. Phenotypic and metabolic analyses were conducted in silenced plants to investigate the specific roles of these enzymes in the biosynthesis of essential amino acids within the plastid. The NbAsp5 silenced plants had no changes in phenotype, exhibiting similar levels of free Asp and glutamate as control plants, but contained diminished levels of asparagine and much higher levels of lysine. In contrast, the suppression of NbPAT led to a severe reduction in growth and strong chlorosis symptoms. NbPAT silenced plants exhibited extremely reduced levels of asparagine and were greatly affected in their phenylalanine metabolism and lignin deposition. Furthermore, NbPAT suppression triggered a transcriptional reprogramming in plastid nitrogen metabolism. Taken together, our results indicate that NbPAT has an overlapping role with NbAsp5 in the biosynthesis of Asp and a key role in the production of phenylalanine for the biosynthesis of phenylpropanoids. The analysis of NbAsp5/NbPAT cosilenced plants highlights the central role of both plastidic aminotransferases in nitrogen metabolism; however, only NbPAT is essential for plant growth and development.

Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

Accumulation of d-leucine, d-allo-isoleucine, and d-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of l-isoleucine to d-allo-isoleucine and d-allo-isoleucine to l-isoleucine. In contrast, the enzyme showed no γ-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 μmol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 μmol·min(-1)·mg(-1), respectively. Hydroxylamine and other inhibitors of pyridoxal 5'-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5'-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.

Novel and recurrent mutations in the TAT gene in Tunisian families affected with Richner–Hanhart Syndrome

Tyrosinemia type II, also designated as oculocutaneous tyrosinemia or Richner–Hanhart syndrome (RHS), is a very rare autosomal recessive disorder. In the present study, we report clinical features and molecular genetic investigation of the tyrosine aminotransferase (TAT) gene in two young patients, both born to consanguineous unions between first-degree cousins. These two unrelated families originated from Northern and Southern Tunisia. The clinical diagnosis was based on the observation of several complications related to Richner–Hanhart syndrome: recurrent eye redness, tearing and burning pain, photophobia, bilateral pseudodendritic keratitis, an erythematous and painful focal palmo-plantar hyperkeratosis and a mild delay of mental development. The diagnosis was confirmed by biochemical analysis. Sequencing of the TAT gene revealed the presence of a previously reported missense mutation (c.452G>A, p.Cys151Tyr) in a Tunisian family, and a novel G duplication (c.869dupG, p.Trp291Leufs*6). Early diagnosis of RHS and protein-restricted diet are crucial to reduce the risk and the severity of long-term complications of hypertyrosinemia such as intellectual disability.

Co-expression of Gamma-Aminobutyrate Aminotransferase and Succinic Semialdehyde Dehydrogenase Genes for the Enzymatic Analysis of Gamma-Aminobutyric Acid in Escherichia Coli

Gamma-aminobutyric acid (GABA) aminotransferase (gabT) and succinic semialdehyde dehydrogenase (gabD) genes from Pseudomonas fluorescens KCCM 12537 were cloned into a single pETDuet-1 vector and co-expressed in Escherichia coli BL21(DE3) simultaneously. The mixture of both enzymes, called GABase, is the key enzyme for the enzymatic analysis of GABA. The molecular mass of the GABA aminotransferase and succinic semialdehyde dehydrogenase were determined to be 52.8 and 46.7 kDa following computations performed with the pI/Mw program, respectively. The GABase activity between pH 6.0 and 9.0 for 24 h at <TEX>$4^{\circ}C$</TEX> remained over 75%, but under pH 6.0 decreased rapidly. The GABase activity between 25 and <TEX>$35^{\circ}C$</TEX> by the treatment at pH 8.6 for 30 min remained over 80%, but over <TEX>$35^{\circ}C$</TEX> decreased rapidly. When the activity against GABA was defined as 100%, the purified GABase activity against 5-aminovaleric acid having a similar structure to GABA showed 47.7% and GABase activity against <TEX>${\beta}$</TEX>-alanine, <TEX>${\varepsilon}$</TEX>-amino-n-caproic acid, <TEX>$_L$</TEX>-ornithine, <TEX>$_L$</TEX>-lysine, and <TEX>$_L$</TEX>-aspartic acid showed between 0.3 to 2.3%. The GABA content was analyzed with this co-expressed GABase, compared with the other GABase which was available commercially. As a result, the content of GABA extracted from brown rice, dark brown rice, and black rice were <TEX>$26.4{\pm}3.5$</TEX>, <TEX>$40.5{\pm}4.7$</TEX> and <TEX>$94.7{\pm}9.3{\mu}g/g$</TEX>, which were similar data of other GABase in the error ranges.

Molecular cloning and overexpression of the tyrosine aminotransferase (TAT) gene leads to increased rosmarinic acid yield in Perilla frutescens

Perilla frutescens is a medicinal plant that produces various bioactive compounds, including unsaturated fatty acids and phenolic compounds such as rosmarinic acid (α-O-caffeoyl-3, 4 -dihydroxyphenylacetic acid, RA). Tyrosine aminotransferase (TAT) catalyzes the first step in the tyrosine-derived branch of RA biosynthesis, and TAT is presumed to have a role in RA accumulation. Here, we report the isolation of full-length TAT cDNA (designated PfTAT) from P. frutescens. Sequence analysis revealed that PfTAT contained 1,535 bp long and an open reading frame of 1,233 bp encoding 411 amino acid residues. Analysis of PfTAT genomic DNA revealed 7 exons and 5 introns. The 5′ flanking sequence of PfTAT was also cloned, and a group of putative cis-acting elements such TATA box, CAAT box, TC-rich repeats and G box were identified. Quantitative real-time PCR analysis indicated that constitutive expression of PfTAT in leaves was much higher than in roots and stems. A vector was constructed containing the PfTAT gene derived by the cauliflower mosaic virus 35S promoter. Transgenic P. frutescens overexpressing PfTAT was obtained with an Agrobacterium tumefaciens-mediated transformation system and overexpression was confirmed by PCR and Southern blot. PfTAT mRNA expression in transgenic plant lines measured by real-time quantitative PCR was 2–3 times greater than PfTAT expression in the untransformed plant line. Also, enhanced gene expression corresponded to significantly increased RA in PfTAT-transgenic lines, as quantified by HPLC. Our data emphasize the importance of PfTAT in the production of RA in P. frutescens.

Roles of YUCCAs in auxin biosynthesis and drought stress responses in plants

Auxin, a plant hormone, plays crucial roles in diverse aspects of plant growth and development reacting to and integrating environmental stimuli. Indole-3-acetic acid (IAA) is the major plant auxin that is synthesized by members of the YUCCA (YUC) family of flavin monooxygenases that catalyse a rate-limiting step. Although the paths to IAA biosynthesis are characterized in Arabidopsis, little is known about the corresponding components in potato. Recently, we isolated eight putative StYUC (Solanum tuberosum YUCCA) genes and five putative tryptophan aminotransferase genes in comparison to those found in Arabidopsis.1 The specific domains of YUC proteins were well conserved in all StYUC amino acid sequences. Transgenic potato (Solanum tuberosum cv. Jowon) overexpressing AtYUC6 showed high-auxin and enhanced drought tolerance phenotypes. The transgenic potatoes also exhibited reduced levels of ROS (reactive oxygen species) compared to control plants. We therefore propose that YUCCA and TAA families in potato would function in the auxin biosynthesis. The overexpression of AtYUC6 in potato establishes enhanced drought tolerance through regulated ROS homeostasis.

Genome-wide identification of the class III aminotransferase gene family in rice and expression analysis under abiotic stress

Aminotransferases are pyridoxal 5′-phosphate-dependent enzymes that play crucial roles in plant growth, development, and responses to abiotic stress. The class III aminotransferase family (ATIII family) is an important subfamily. However, no characterization of rice ATIII genes has been previously reported. Using available rice genome sequence information, we identified 12 japonica and 13 indicaATIII genes that were randomly localized on chromosomes 2, 3, 4, 5, 7, 8, and 11. Information provided by the Plant Genome Duplication Database revealed that four japonica and four indicaATIII genes are the results of segmental duplications, and two japonica and six indica genes resulted from tandem duplications. A phylogenetic analysis of the ATIII genes in japonica, indica and Arabidopsis enabled the classification of the genes into six different groups, and the characteristics were established before the monocot-dicot and japonica–indica split. An analysis of the Ka/Ks, divergence time and average indel length suggested the diverse selection styles of the duplicated gene pairs. Gene structure and motif analyses revealed that the ATIII gene family has experienced extensive divergence. Real-time PCR was performed to examine the expression pattern of the japonicaATIII genes in response to various abiotic stresses including drought, salt, and cold. The results suggested that most of the genes were differentially up- or down-regulated in rice seedlings in response to at least one stress factor, which indicates the key role of the rice ATIII gene family in responding to abiotic stresses. These results provide a basis for elucidating the roles of the ATIII genes and their further functional analysis under abiotic stresses.

BCAT2 regulates BCAA metabolic fate during adipogenesis

Adipogenesis is a controlled differentiation process regulated by a network of transcription factors that include C/EBPβ and PPARγ. PPARγ binds to the vicinity of &gt;;5000 genes through the different stages of differentiation (Nielsen et al, Genes Dev 2008(22):2953–2967). An analysis of these genes revealed that one of them is the mitochondrial branched‐chain aminotransferase (BCAT2) gene. BCAT2 catalyze the first enzymatic step of the branched chain amino acids (BCAA) catabolism. However, the role of BCTA2 during adipogenesis is barely known. Thus, we have evaluated BCAT2 expression and the oxidation, incorporation to lipids or proteins of the three BCAA, leucine, isoleucine and valine, during adipogenesis. Undifferentiated 3T3‐L1 did not express BCAT2 but both its mRNA and protein levels were gradually increased after induction of differentiation, reaching their maximum level at day 12. Regarding BCAA metabolic fate, BCAA were incorporated into protein when BCAT2 was not present, and were mainly oxidized and incorporated into lipids as BCAT2 increased during the differentiation process. Interestingly, leucine was mainly oxidized in adipocytes from lean mice and this was decreased in adipocytes from obese mice. These data demonstrate that BCAT2 regulates the metabolic fate of BCAA during adipogenesis and establish a process that is altered during obesity. Supported by CONACYT‐155949 to LGN.

Long-Term Renal Allograft Survival After Posttransplantation Diagnosis of Primary Hyperoxaluria

Primary hyperoxaluria (PH) is a rare metabolic disorder with autosomal recessive inheritance characterized by excessive production and systemic deposition of oxalate. Unfortunately, the diagnosis in some patients is not made until after kidney transplantation. These grafts are nearly always lost unless the patient receives a liver transplant to correct the underlying defect in oxalate metabolism. We describe a patient who developed extensive oxalate deposition within her transplanted kidney necessitating a return to hemodialysis (HD). After treatment with pyridoxine, high-flux dialysis, and substitution of sirolimus for Cyclosporin A, she ceased dialysis and maintained stable allograft function. A 47-year-old woman was admitted for a deceased-donor kidney transplantation in 2006 with kidney failure attributed to reflux nephropathy and a history of traumatic nephrectomy during childhood. She underwent peritoneal dialysis before transitioning to HD for 3 years. Her medical history included painful peripheral neuropathy and small-joint polyarthropathy characterized by subperiosteal erosions. Surgery was uncomplicated with immediate graft function. Immunosuppression consisted of induction with basiliximab and maintenance with prednisolone, mycophenolate mofetil, and cyclosporin. Kidney biopsies performed on days 7 and 11 after transplantation, for an unexplained decline in renal function, showed changes of mild acute tubular necrosis. A further decline in kidney function on day 22 prompted another biopsy that demonstrated extensive deposition of calcium oxalate crystals with associated tubular damage (Fig. 1).FIGURE 1: Kidney biopsy at 3 weeks after transplantation showing extensive oxalate deposition. A, hematoxylin and eosin. Original magnification, ×100. B, under polarized light. Original magnification, ×400.Given the likely diagnosis of PH, daily high-flux HD, pyridoxine (1), dithiazide, magnesium and phosphate supplements, and a low-oxalate diet were commenced (2). Urinary oxalate excretion was elevated at 1.2 mM/day (normal, <0.6 mM/day). Cyclosporin was ceased given the association with hyperoxaluria and hypocitraturia, renal calcium-oxalate deposition (3), and poor kidney transplantation outcomes (4, 5). The serum creatinine stabilized at 150 μM/L after 5 weeks. Rapid and extensive deposition of oxalate within the small joints of the hand resulted in a gross deforming polyarthropathy and, within the bone marrow, led to refractory anemia. The diagnosis of type-1 hyperoxaluria was confirmed by genetic testing where a mutation in the alanine:glyoxylate aminotransferase (AGT) gene (homozygous 508A missense mutation, Gly170Arg) was identified. Subsequent family screening identified several other family members with the same AGT gene mutation. A protocol kidney biopsy at 3 months showed acute T-cell–mediated rejection (Grade IB) and persistent oxalate deposition. The rejection resolved after three doses of methylprednisolone and addition of sirolimus. Eight months after transplantation, she had a prolonged hospital admission with an ischemic toe requiring amputation, Clostridium difficile colitis, and extensive deep-vein thrombosis. A rise in serum creatinine prompted a biopsy that demonstrated T-cell–mediated rejection (Grade IA) and extensive oxalate deposition. Because of her multiple medical problems, the rejection episode was not treated. HD was recommenced and immunosuppression (prednisolone, mycophenolate mofetil, and low-dose sirolimus; levels 4–6 ng/mL) continued to preserve residual graft function and aid in oxalate clearance. While on HD, she maintained a daily urine output of 1.5 to 2 L/day. After 18 months of HD, the patient’s predialysis serum creatinine began to decline. HD was slowly weaned, and she now maintains a serum creatinine level of 175 μmol/L 5 years later. PH type 1 is caused by a deficiency of the liver-specific peroxisomal enzyme AGT, and PH type 2 is caused by a deficiency of the cytosolic/mitochondrial enzyme glyoxylate/hydroxypyruvate reductase (2). PH type 3 has only recently been described and is caused by the loss of function of the mitochondrial 4-hydroxy-2-oxoglutarate aldolase enzyme (6). Clinical presentation of PH is heterogenous, often leading to delayed diagnosis. Prognosis and treatment options vary according to the type of PH (reviewed in Hoppe [7]). Liver transplantation with or without kidney transplantation is the only cure for PH type 1. Transplantation outcomes in children and adults with PH demonstrate better kidney graft survival in patients with combined liver-kidney transplantation (8, 9) than in those with kidney transplantation alone. However, in patients with PH type 1 with the homozygous G170R mutation of alanine-glyoxalate aminotransferase and who are responsive to pyridoxine treatment, kidney transplantation alone may be considered (10). Late diagnosis of PH is not uncommon, and it is important to secure the genetic mutation involved as soon as possible. Our patient has a mutation that responded to pyridoxine treatment (11). This case also illustrates that it is possible to preserve kidney allograft function in some patients with PH. Furthermore, replacing Cyclosporin A with a mammalian target of rapamycin inhibitor may reduce oxalate deposition and contribute to improved graft outcomes (12). Veena Roberts 1 Sid V. Rajakumar1 Sandra Crikis2 Karen M. Dwyer1 Prudence A. Hill3 David J. Goodman1 1Department of Nephrology St Vincent’s Hospital Melbourne Melbourne, Australia 2Department of Nephrology Western Health Fitzroy, Australia 3Department of Anatomical Pathology St Vincent’s Hospital Melbourne Melbourne, Australia

Iron-biofortification in rice by the introduction of three barley genes participated in mugineic acid biosynthesis with soybean ferritin gene

Iron deficiency is a serious problem around the world, especially in developing countries. The production of iron-biofortified rice will help ameliorate this problem. Previously, expression of the iron storage protein, ferritin, in rice using an endosperm-specific promoter resulted in a two-fold increase in iron concentration in the resultant transgenic seeds. However, further over expression of ferritin did not produce an additional increase in the seed iron concentration, and symptoms of iron deficiency were noted in the leaves of the transgenic plants. In the present study, we aimed to further increase the iron concentration in rice seeds without increasing the sensitivity to iron deficiency by enhancing the uptake and transport of iron via a ferric iron chelator, mugineic acid. To this end, we introduced the soybean ferritin gene (SoyferH2) driven by two endosperm-specific promoters, along with the barley nicotianamine synthase gene (HvNAS1), two nicotianamine aminotransferase genes (HvNAAT-A and -B), and a mugineic acid synthase gene (IDS3) to enhance mugineic acid production in rice plants. A marker-free vector was utilized as a means of increasing public acceptance. Representative lines were selected from 102 transformants based on the iron concentration in polished seeds and ferritin accumulation in the seeds. These lines were grown in both commercially supplied soil (iron-sufficient conditions) and calcareous soil (iron-deficient conditions). Lines expressing both ferritin and mugineic acid biosynthetic genes showed signs of iron-deficiency tolerance in calcareous soil. The iron concentration in polished T3 seeds was increased by 4 and 2.5 times, as compared to that in non-transgenic lines grown in normal and calcareous soil, respectively. These results indicate that the concomitant introduction of the ferritin gene and mugineic acid biosynthetic genes effectively increased the seed iron level without causing iron sensitivity under iron-limited conditions.

Successful mouse hepatocyte culture with sandwich collagen gel formation

PurposePrimary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking.MethodsPrimary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied.ResultsAfter 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture.ConclusionThe collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.

Engineering of Corynebacterium glutamicum for High-Yieldl-Valine Production under Oxygen Deprivation Conditions

We previously demonstrated efficient L-valine production by metabolically engineered Corynebacterium glutamicum under oxygen deprivation. To achieve the high productivity, a NADH/NADPH cofactor imbalance during the synthesis of l-valine was overcome by engineering NAD-preferring mutant acetohydroxy acid isomeroreductase (AHAIR) and using NAD-specific leucine dehydrogenase from Lysinibacillus sphaericus. Lactate as a by-product was largely eliminated by disrupting the lactate dehydrogenase gene ldhA. Nonetheless, a few other by-products, particularly succinate, were still produced and acted to suppress the L-valine yield. Eliminating these by-products therefore was deemed key to improving theL-valine yield. By additionally disrupting the phosphoenolpyruvate carboxylase gene ppc, succinate production was effectively suppressed, but both glucose consumption and L-valine production dropped considerably due to the severely elevated intracellular NADH/NAD(+) ratio. In contrast, this perturbed intracellular redox state was more than compensated for by deletion of three genes associated with NADH-producing acetate synthesis and overexpression of five glycolytic genes, including gapA, encoding NADH-inhibited glyceraldehyde-3-phosphate dehydrogenase. Inserting feedback-resistant mutant acetohydroxy acid synthase and NAD-preferring mutant AHAIR in the chromosome resulted in higher L-valine yield and productivity. Deleting the alanine transaminase gene avtA suppressed alanine production. The resultant strain produced 1,280 mM L-valine at a yield of 88% mol mol of glucose(-1) after 24 h under oxygen deprivation, a vastly improved yield over our previous best.

Biochemical Properties and Crystal Structure of a β-Phenylalanine Aminotransferase from Variovorax paradoxus

By selective enrichment, we isolated a bacterium that can use β-phenylalanine as a sole nitrogen source. It was identified by 16S rRNA gene sequencing as a strain of Variovorax paradoxus. Enzyme assays revealed an aminotransferase activity. Partial genome sequencing and screening of a cosmid DNA library resulted in the identification of a 1,302-bp aminotransferase gene, which encodes a 46,416-Da protein. The gene was cloned and overexpressed in Escherichia coli. The recombinant enzyme was purified and showed a specific activity of 17.5 U mg(-1) for (S)-β-phenylalanine at 30°C and 33 U mg(-1) at the optimum temperature of 55°C. The β-specific aminotransferase exhibits a broad substrate range, accepting ortho-, meta-, and para-substituted β-phenylalanine derivatives as amino donors and 2-oxoglutarate and pyruvate as amino acceptors. The enzyme is highly enantioselective toward (S)-β-phenylalanine (enantioselectivity [E], >100) and derivatives thereof with different substituents on the phenyl ring, allowing the kinetic resolution of various racemic β-amino acids to yield (R)-β-amino acids with >95% enantiomeric excess (ee). The crystal structures of the holoenzyme and of the enzyme in complex with the inhibitor 2-aminooxyacetate revealed structural similarity to the β-phenylalanine aminotransferase from Mesorhizobium sp. strain LUK. The crystal structure was used to rationalize the stereo- and regioselectivity of V. paradoxus aminotransferase and to define a sequence motif with which new aromatic β-amino acid-converting aminotransferases may be identified.

Primary hyperoxaluria type 1 and brachydactyly mental retardation syndrome caused by a novel mutation in AGXT and a terminal deletion of chromosome 2

Primary hyperoxaluria type 1 (PH1) is an autosomal recessive disorder caused by mutations in the alanine:glyoxylate aminotransferase (AGXT) gene, located on chromosome 2q37. Mutant AGXT leads to excess production and excretion of oxalate, resulting in accumulation of calcium oxalate in the kidney, and progressive loss of renal function. Brachydactyly mental retardation syndrome (BDMR) is an autosomal dominant disorder, caused by haploinsufficiency of histone deacetylase 4 (HDAC4), also on chromosome 2q37. It is characterized by skeletal abnormalities and developmental delay. Here, we report on a girl who had phenotypes of both PH1 and BDMR. PCR-sequencing of the coding regions of AGXT showed a novel missense mutation, c.32C>G (p.Pro11Arg) inherited from her mother. Functional analyses demonstrated that it reduced the enzymatic activity to 31% of the wild-type and redirected some percentage of the enzyme away from the peroxisome. Microsatellite and array-CGH analyses indicated that the proband had a paternal de novo telomeric deletion of chromosome 2q, which included HDAC4. To our knowledge, this is the first report of PH1 and BDMR, with a novel AGXT mutation and a de novo telomeric deletion of chromosome 2q.

Functional validation of Capsicum frutescens aminotransferase gene involved in vanillylamine biosynthesis using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures

Capsaicinoid biosynthesis involves the participation of two substrates viz. vanillylamine and C9–C11 fatty acid moieties. Vanillylamine which is a derivative of vanillin is synthesized through a transaminase reaction in the phenylpropanoid pathway of capsaicinoid synthesis. Here we report the functional validation of earlier reported putative aminotransferase gene for vanillylamine biosynthesis in heterologous system using Agrobacterium mediated genetic transformation studies in Nicotiana tabacum and Capsicum frutescens calli cultures. Molecular analysis tools comprising PCR and Southern blot analysis have shown the integration of the foreign gene in N. tabacum and C. frutescens calli cultures. The study shows the production of vanillylamine in transformed N. tabacum callus cultures and also the reduction of vanillylamine production when whole gene based antisense binary vector construct was used in transformation of C. frutescens callus cultures. Vanillylamine production, aminotransferase assay with Western blot analysis for crude proteins of transformants established the production of putative aminotransferase (pAMT) protein in alternate plant. The result is a clear evidence of involvement of the reported putative aminotransferase responsible for vanillylamine biosynthesis in capsaicinoid biosynthesis pathway, confirming the gene function through functional validation.

Overexpression of human antiquitin in E. coli: Enzymatic characterization of twelve ALDH7A1 missense mutations associated with pyridoxine-dependent epilepsy

Pyridoxine dependent epilepsy is an autosomal recessive disorder characterized by early onset seizures responsive to pyridoxine and caused by a defect in the α-aminoadipic semialdehyde dehydrogenase (antiquitin) gene (ALDH7A1). In order to characterize the effects of a series of twelve disease-associated ALDH7A1 missense mutations on antiquitin activity, we generated the mutations in a recombinant human antiquitin cDNA and expressed them in Escherichia coli. We developed an automated spectrophotometric assay of antiquitin enzymatic activity using the natural substrate α-aminoadipic semialdehyde. The substrate was generated using a recombinant lysine aminotransferase gene (lat) from Streptomyces clavuligerus. In the E. coli expression system all the mutants were stably expressed but lacked enzymatic activity. This is consistent with pathogenicity of these mutations in vivo.

Neuropathies associated with oxaliplatin therapy

Neuropathies associated with oxaliplatin therapy