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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the indica rice (Oryza sativa L.) a cDNA was characterized that encoded OsAKT1 homologous to inward-rectifying potassium channels of the AKT/KAT subfamily. Transcript analysis located OsAKT1 predominantly in roots with low abundance in leaves. Cell-specificity of OsAKT expression was analyzed by in situ hybridizations. In roots, strongest signals were localized to the epidermis and the endodermis, whereas lower transcript levels were detected in cells of the vasculature and the cortex. In leaves, expression was detected in xylem parenchyma, phloem, and mesophyll cells. Transcriptional regulation and cell specificity of OsAKT1 during salt stress was compared in rice lines showing different salinity tolerance. In the salt-tolerant, sodium-excluding varieties Pokkali and BK, OsAKT1 transcripts disappeared from the exodermis in plants treated with 150 mM NaCl for 48 h but OsAKT1 transcription was not repressed in these cells in the salt-sensitive, sodium-accumulating variety IR29. Significantly, all lines were able to maintain potassium levels under sodium stress conditions, while sodium concentrations in the leaves of IR29 increased 5-10-fold relative to the sodium concentration in BK or Pokkali. The divergent, line-dependent and salt-dependent, regulation of this channel does not significantly affect potassium homeostasis under salinity stress. Rather, repression in Pokkali/BK and lack of repression in IR29 correlate with the overall tolerance character of these lines.
Plant Mol Biol 2003 Jan
PMID:Salinity stress-tolerant and -sensitive rice (Oryza sativa L.) regulate AKT1-type potassium channel transcripts differently. 1260 92

The biosynthesis of polyhydroxyalkanoates (PHAs) was studied, for the first time, in the thermophilic bacterium Thermus thermophilus. Using sodium gluconate (1.5% w/v) or sodium octanoate (10 mM) as sole carbon sources, PHAs were accumulated to approximately 35 or 40% of the cellular dry weight, respectively. Gas chromatographic analysis of PHA isolated from gluconate-grown cells showed that the polyester (Mw: 480,000 g mol(-1)) was mainly composed of 3-hydroxydecanoate (3HD) with a molar fraction of 64%. In addition, 3-hydroxyoctanoate (3HO), 3-hydroxyvalerate (3HV) and 3-hydroxybutyrate (3HB) occurred as constituents. In contrast, the polyester (Mw: 391,000 g mol(-1)) from octanoate-grown cells was composed of 24.5 mol% 3HB, 5.4 mol% 3HO, 12.3 mol% 3-hydroxynonanoate (3HN), 14.6 mol% 3HD, 35.4 mol% 3-hydroxyundecanoate (3HUD) and 7.8 mol% 3-hydroxydodecanoate (3HDD). Activities of PHA synthase, a beta-ketothiolase and an NADPH-dependent reductase were detected in the soluble cytosolic fraction obtained from gluconate-grown cells of T. thermophilus. The soluble PHA synthase was purified 4271-fold with 8.5% recovery from gluconate-grown cells, presenting a Km of 0.25 mM for 3HB-CoA. The optimal temperature of PHA synthase activity was about 70 degrees C and acts optimally at pH near 7.3. PHA synthase activity was inhibited 50% with 25 microM CoA and lost all of its activity when it was treated with alkaline phosphatase. T. thermophilus PHA synthase, in contrary to other reported PHA synthases did not exhibit a lag phase on its kinetics, when low concentration of the enzyme was used. Incubation of PHA synthase with 1 mM N-ethyl-maleimide inhibits the enzyme 56%, indicating that cysteine might be involved in the catalytic site of the enzyme. Acetyl phosphate (10 mM) activated both the native and the dephosphorylated enzyme. A major protein (55 kDa) was detected by SDS-PAGE. When a partially purified preparation was analyzed on native PAGE the major band exhibiting PHA synthase activity was eluted from the gel and analyzed further on SDS-PAGE, presenting the first purification of a PHA synthase from a thermophilic microorganism.
Mol Cell Biochem 2003 Dec
PMID:Polyhydroxyalkanoate (PHA) biosynthesis in Thermus thermophilus: purification and biochemical properties of PHA synthase. 1467 96

A 19-month-old boy with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency, a defect of isoleucine degradation, had cognitive and motor development delay, spastic diplegia, dysmorphism, and occipital periventricular white matter lesions on MRI scan of the brain. The urinary accumulation of the isoleucine metabolites 2-methyl-3-hydroxybutyrate and tiglylglycine was only moderate under basal conditions. These abnormalities became more pronounced after a 100mg/kg oral isoleucine challenge. Enzyme studies showed a markedly decreased activity of MHBD in fibroblasts and lymphocytes. Sequence analysis of the involved X-chromosome gene (HADH2), revealed the presence of 364C -->G mutation in the patient. His mother was heterozygous for the 364C-->G mutation, whereas the mutation was not found in the other members of the family (father, brother, and sister). This report indicates that an enzyme defect in the metabolism of branched-chain fatty acid oxidation and isoleucine may present features resembling sequelae of neonatal hypoxic-ischemic brain injury. All patients with MHBD deficiency identified so far are characterized by a neurologic phenotype rather than ketoacidotic attacks, unlike patients with the related isoleucine defect beta-ketothiolase deficiency.
Mol Genet Metab 2004 Apr
PMID:Spastic diplegia and periventricular white matter abnormalities in 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, a defect of isoleucine metabolism: differential diagnosis with hypoxic-ischemic brain diseases. 1505 17

Kynurenic acid (KYNA) can act as an endogenous modulator of excitatory neurotransmission and has been implicated in the pathogenesis of several neurological and psychiatric diseases. To evaluate its role in the brain, we disrupted the murine gene for kynurenine aminotransferase II (KAT II), the principal enzyme responsible for the synthesis of KYNA in the rat brain. mKat-2(-/-) mice showed no detectable KAT II mRNA or protein. Total brain KAT activity and KYNA levels were reduced during the first month but returned to normal levels thereafter. In contrast, liver KAT activity and KYNA levels in mKat-2(-/-) mice were decreased by >90% throughout life, though no hepatic abnormalities were observed histologically. KYNA-associated metabolites kynurenine, 3-hydroxykynurenine, and quinolinic acid were unchanged in the brain and liver of knockout mice. mKat-2(-/-) mice began to manifest hyperactivity and abnormal motor coordination at 2 weeks of age but were indistinguishable from wild type after 1 month of age. Golgi staining of cortical and striatal neurons revealed enlarged dendritic spines and a significant increase in spine density in 3-week-old mKat-2(-/-) mice but not in 2-month-old animals. Our results show that gene targeting of mKat-2 in mice leads to early and transitory decreases in brain KAT activity and KYNA levels with commensurate behavioral and neuropathological changes and suggest that compensatory changes or ontogenic expression of another isoform may account for the normalization of KYNA levels in the adult mKat-2(-/-) brain.
Mol Cell Biol 2004 Aug
PMID:Biochemical and phenotypic abnormalities in kynurenine aminotransferase II-deficient mice. 1528 94

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates (HAs) synthesised by numerous bacteria as intracellular carbon and energy storage compounds which accumulate as granules in the cytoplasm of the cells. The biosynthesis of PHAs, in the thermophilic bacterium T. thermophilus grown in a mineral medium supplemented with sodium gluconate as sole carbon source has been recently reported. Here, we report the purification at apparent homogeneity of a beta-ketoacyl-CoA thiolase from T. thermophilus, the first enzyme of the most common biosynthetic pathway for PHAs. B-Ketoacyl-CoA thiolase appeared as a single band of 45.5-kDa molecular mass on SDS/PAGE. The enzyme was purified 390-fold with 7% recovery. The native enzyme is a multimeric protein of a molecular mass of approximately of 182 kDa consisting of four identical subunits of 45.5 kDa, as identified by an in situ renaturation experiment on SDS-PAGE. The enzyme exhibited an optimal pH of approximately 8.0 and highest activity at 65 degrees C for both direction of the reaction. The thiolysis reaction showed a substrate inhibition at high concentrations; when one of the substrates (acetoacetyl CoA or CoA) is varied, while the concentrations of the second substrates (CoA or acetoacetyl CoA respectively) remain constant. The initial velocity kinetics showed a pattern of a family of parallel lines, which is in accordance with a ping-pong mechanism. beta-Ketothiolase had a relative low Km of 0.25 mM for acetyl-CoA and 11 microM and 25 microM for CoA and acetoacetyl-CoA, respectively. The enzyme was inhibited by treatment with 1 mM N-ethylmaleimide either in the presence or in the absence of 0.5 mM of acetyl-CoA suggesting that possibly a cysteine is located at/or near the active site of beta-ketothiolase.
Mol Cell Biochem 2005 Jan
PMID:A thermostable beta-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: purification and biochemical properties. 1578 14

Long-chain l-3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency has been included in the routine neonatal screening program by the German screening commission. As tandem mass spectrometry (TMS) does not discriminate between the different defects of the mitochondrial trifunctional protein (MTP) screening for isolated LCHAD deficiency includes the detection of long-chain 3-ketoacyl-CoA thiolase and complete MTP deficiencies as well. We identified 11 patients with abnormalities of the MTP out of 1.2 million newborns screened in our laboratory during the last 6 years. Treatment was started on the day the screening result was obtained (day 3 to day 9 of life). Seven of these newborns developed satisfactorily during an observation period of up to 64 months. They had isolated LCHAD deficiency, four of them caused by the typical mutation (1528 G>C), three others had no molecular genetic analysis done or were shown to have previously unknown mutations. Four children did not survive, two of them showing complete deficiency of MTP and two showing deficiency of long-chain 3-ketoacyl-CoA thiolase. We conclude that, despite the rarity of the disease, screening for MTP deficiencies is justified based on the following criteria: improved quality of life for patients with isolated LCHAD deficiency, absence of stigmatisation of babies showing mild variants without necessity of treatment, no significant increase of the total number of false positive screening results, no false negative results to our knowledge. Finally, extension of analysis to MTP deficiencies is achieved without additional costs for screening laboratories already using TMS.
Mol Genet Metab 2005 Jun
PMID:Neonatal screening for defects of the mitochondrial trifunctional protein. 1589 54

Eukaryotic thiolases are essential enzymes located in three different compartments (peroxisome, mitochondrion, and cytosol) that can display catabolic or anabolic functions. They are responsible for the thiolytic cleavage of oxidized acyl-CoA (thiolase I; EC 2.3.1.16) and the synthesis or degradation of acetoacetyl-CoA (thiolase II; EC 2.3.1.9). Phylogenetic analysis of eukaryotic thiolase sequences showed that they form six distinct clusters, one of them highly divergent, which are in good correlation with their class and subcellular location. When analyzed together with a representative sample of prokaryotic thiolases, all eukaryotic thiolase groups emerged close to proteobacterial sequences. Metazoan cytosolic thiolase II was related to alpha-proteobacterial sequences, suggesting a mitochondrial origin. Unexpectedly, cytosolic thiolases from green plants and fungi as well as at least one member of all eukaryotic peroxisomal and mitochondrial thiolases had delta-proteobacteria as closest relatives. Our analysis suggests that these eukaryotic peroxisomal and mitochondrial thiolases may have been acquired from delta-proteobacteria prior to the ancestor of all known eukaryotes.
J Mol Evol 2005 Jul
PMID:Phylogenetic analysis of eukaryotic thiolases suggests multiple proteobacterial origins. 1598 Sep 57

Since a few years ESI-MS/MS has been employed for the simultaneous detection of a wide range of inborn errors of metabolism. The screening center North at the Hamburg University Medical Center processes 40-50,000 samples per year. To assess current developments in neonatal screening, the Northern German Working Group on Neonatal Screening consisting of health care providers, metabolic centers, and screening laboratories was founded. Based on current literature and experience four categories of diseases were established. The first three categories were recommended for screening under constant scientific evaluation, while glutaric aciduria II, beta-ketothiolase deficiency, short-chain acylCoA dehydrogenase deficiency, and homocystinuria were not included in the screening program. In contrast, general screening for phenylketonuria (PKU) remains undisputed and MS/MS screening reduced false positives by simultaneously detecting phenylalanine and tyrosine. Recently, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4))-sensitive PKU has been discovered. We were able to demonstrate that BH(4) treatment without dietary restrictions may be sufficient for certain BH(4)-responsive PKU patients. In general, MS/MS provides a potential to rapidly screen for a wide variety of rare metabolic disorders but a close cooperation between scientists and metabolic doctors is required to constantly evaluate results in terms of improving the outcome of patients.
Mol Nutr Food Res 2006 Apr
PMID:Evaluation of electrospray-tandem mass spectrometry for the detection of phenylketonuria and other rare disorders. 1659 11

Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.
J Mol Biol 2006 Jun 02
PMID:The crystal structure of a plant 3-ketoacyl-CoA thiolase reveals the potential for redox control of peroxisomal fatty acid beta-oxidation. 1663 Jun 29

Agrobacterium tumefaciens-mediated random mutagenesis was used to generate insertional mutants of the fungus Leptosphaeria maculans. Of 91 transformants screened, only one (A3) produced lesions of reduced size on cotyledons of canola (Brassica napus). Genes flanking the T-DNA insertion had the best matches to an alcohol dehydrogenase class 4 (ADH4)-like gene (Adh4L) and a 3-ketoacyl-CoA thiolase gene (Thiol) and were expressed in mutant A3 in vitro and in planta at significantly higher levels than in the wild type. This is the first report of a T-DNA insertion in fungi causing increased gene expression. Transformants of the wild-type isolate expressing both Adh4L and Thiol under the control of a heterologous promoter had similar pathogenicity to mutant A3. Ectopic expression of only thiolase resulted in loss of pathogenicity, suggesting that thiolase overexpression was primarily responsible for the reduced pathogenicity of the A3 isolate. The thiolase gene encoded a functional protein, as shown by assays in which a nontoxic substrate (2, 4 dichlorophenoxybutyric acid) was converted to a toxic product. The use of a translational fusion with a reporter gene showed thiolase expressed in organelles that are most likely peroxisomes.
Mol Plant Microbe Interact 2006 Jun
PMID:Overexpression of a 3-ketoacyl-CoA thiolase in Leptosphaeria maculans causes reduced pathogenicity on Brassica napus. 1677 92


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