Publications by authors named "Birgit Andersen"

23 Publications

  • Page 1 of 1

Novel Homozygous Truncating Variant Widens the Spectrum of Early-Onset Multisystemic SYNE1 Ataxia.

Cerebellum 2021 Jul 28. Epub 2021 Jul 28.

Department of Neurology, University of Copenhagen, Rigshospitalet, Inge Lehmanns Vej 8, 2100, Copenhagen, Denmark.

Pathogenic variants in the SYNE1 gene are associated with a phenotypic spectrum spanning from late-onset, slowly progressive, relatively pure ataxia to early-onset, fast progressive multisystemic disease. Since its first description in 2007 as an adult-onset ataxia in French Canadian families, subsequent identification of patients worldwide has widened the clinical spectrum and increased the number of identified pathogenic variants. We report a 20-year-old Faroese female with early-onset progressive gait problems, weakness, dysphagia, slurred speech, orthostatic dizziness, and urge incontinence. Neurological examination revealed mild cognitive deficits, dysarthria, broken slow pursuit, hypometric saccades, weakness with spasticity, hyperreflexia, absent ankle reflexes, ataxia, and wide-based, spastic gait. Magnetic resonance imaging displayed atrophy of the cerebellum, brainstem, and spinal cord. Severely prolonged central motor conduction time and lower motor neuron involvement was demonstrated electrophysiologically. Fluorodeoxyglucose-positron emission tomography (FDG-PET) scan showed hypometabolism of the cerebellum and right frontal lobe. Muscle biopsy revealed chronic neurogenic changes and near-absent immunostaining for Nesprin-1. Next-generation sequencing revealed a previously undescribed homozygous truncating, likely pathogenic variant in the SYNE1 gene. The patient's mother and paternal grandfather were heterozygous carriers of the variant. Her father's genotype was unobtainable. We expand the list of likely pathogenic variants in SYNE1 ataxia with a novel homozygous truncating variant with proximity to the C-terminus and relate it to a phenotype comprising early-onset cerebellar deficits, upper and lower motor neuron involvement and cognitive deficits. Also, we report novel findings of focally reduced frontal lobe FDG-PET uptake and motor evoked potential abnormalities suggestive of central demyelination.
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http://dx.doi.org/10.1007/s12311-021-01308-wDOI Listing
July 2021

Proteomics insights into the responses of Saccharomyces cerevisiae during mixed-culture alcoholic fermentation with Lachancea thermotolerans.

FEMS Microbiol Ecol 2019 09;95(9)

Department of Food Science, University of Copenhagen, Rolighedsvej 26, 1958 Frederiksberg C, Denmark.

The response of Saccharomyces cerevisiae to cocultivation with Lachancea thermotolerans during alcoholic fermentations has been investigated using tandem mass tag (TMT)-based proteomics. At two key time-points, S. cerevisiae was sorted from single S. cerevisiae fermentations and from mixed fermentations using flow cytometry sorting. Results showed that the purity of sorted S. cerevisiae was above 96% throughout the whole mixed-culture fermentation, thereby validating our sorting methodology. By comparing protein expression of S. cerevisiae with and without L. thermotolerans, 26 proteins were identified as significantly regulated proteins at the early death phase (T1), and 32 significantly regulated proteins were identified at the late death phase (T2) of L. thermotolerans in mixed cultures. At T1, proteins involved in endocytosis, increasing nutrient availability, cell rescue and resistance to stresses were upregulated, and proteins involved in proline synthesis and apoptosis were downregulated. At T2, proteins involved in protein synthesis and stress responses were up- and downregulated, respectively. These data indicate that S. cerevisiae was stressed by the presence of L. thermotolerans at T1, using both defensive and fighting strategies to keep itself in a dominant position, and that it at T2 was relieved from stress, perhaps increasing its enzymatic machinery to ensure better survival.
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http://dx.doi.org/10.1093/femsec/fiz126DOI Listing
September 2019

Quantitative Proteomics Analysis of Barley-Based Liquid Feed and the Effect of Protease Inhibitors and NADPH-Dependent Thioredoxin Reductase/Thioredoxin (NTR/Trx) System.

J Agric Food Chem 2019 Jun 28;67(22):6432-6444. Epub 2019 May 28.

Agricultural and Environmental Proteomics, Department of Systems Biology , Technical University of Denmark , Søltofts Plads, Building 224 , DK-2800 Kgs. Lyngby , Denmark.

Liquid feeding strategies have been devised with the aim of enhancing grain nutrient availability for livestock. It is characterized by a steeping/soaking period that softens the grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples supplemented with either protease inhibitors or NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in the water-extractable fraction of feed containing protease inhibitors, illustrating significant inhibition of proteolytic activities arising during the steeping period. Application of functional NTR/Trx to liquid feed reductively increased the solubility of known and potentially new Trx-target proteins, e.g., outer membrane protein X, and their susceptibility to proteolysis. Thus, the NTR/Trx system exhibits important potential as a feed additive to enhance nutrient digestibility in monogastric animals.
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http://dx.doi.org/10.1021/acs.jafc.9b01708DOI Listing
June 2019

High-throughput analysis of amino acids in plant materials by single quadrupole mass spectrometry.

Plant Methods 2018 19;14. Epub 2018 Jan 19.

Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.

Background: The amino acid profile of plants is an important parameter in assessments of their growth potential, resource-use efficiency and/or quality as food and feed. Screening studies may involve large number of samples but the classical amino acid analysis is limited by the fact that it is very time consuming with typical chromatographic run times of 70 min or more.

Results: We have here developed a high-throughput method for analysis of amino acid profiles in plant materials. The method combines classical protein hydrolysis and derivatization with fast separation by UHPLC and detection by a single quadrupole (QDa) mass spectrometer. The chromatographic run time is reduced to 10 min and the precision, accuracy and sensitivity of the method are in line with other recent methods utilizing advanced and more expensive mass spectrometers. The sensitivity of the method is at least a factor 10 better than that of methods relying on detection by fluorescence or UV. It is possible to downscale sample size to 20 mg without compromising reproducibility, which makes the method ideal for analysis of very small sample amounts.

Conclusion: The developed method allows high-throughput analysis of amino acid profiles in plant materials. The analysis is robust and accurate as well as compatible with both free amino acids and protein hydrolysates. The QDa detector offers high sensitivity and accuracy, while at the same time being relatively simple to operate and cheap to purchase, thus significantly reducing the overall analytical costs compared to methods based on more advanced mass spectrometers.
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http://dx.doi.org/10.1186/s13007-018-0277-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774165PMC
January 2018

Investigation of the indigenous fungal community populating barley grains: Secretomes and xylanolytic potential.

J Proteomics 2017 10 18;169:153-164. Epub 2017 Mar 18.

Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark. Electronic address:

The indigenous fungal species populating cereal grains produce numerous plant cell wall-degrading enzymes including xylanases, which could play important role in plant-pathogen interactions and in adaptation of the fungi to varying carbon sources. To gain more insight into the grain surface-associated enzyme activity, members of the populating fungal community were isolated, and their secretomes and xylanolytic activities assessed. Twenty-seven different fungal species were isolated from grains of six barley cultivars over different harvest years and growing sites. The isolated fungi were grown on medium containing barley flour or wheat arabinoxylan as sole carbon source. Their secretomes and xylanase activities were analyzed using SDS-PAGE and enzyme assays and were found to vary according to species and carbon source. Secretomes were dominated by cell wall degrading enzymes with xylanases and xylanolytic enzymes being the most abundant. A 2-DE-based secretome analysis of Aspergillus niger and the less-studied pathogenic fungus Fusarium poae grown on barley flour and wheat arabinoxylan resulted in identification of 82 A. niger and 31 F. poae proteins many of which were hydrolytic enzymes, including xylanases.

Biological Significance: The microorganisms that inhabit the surface of cereal grains are specialized in production of enzymes such as xylanases, which depolymerize plant cell walls. Integration of gel-based proteomics approach with activity assays is a powerful tool for analysis and characterization of fungal secretomes and xylanolytic activities which can lead to identification of new enzymes with interesting properties, as well as provide insight into plant-fungal interactions, fungal pathogenicity and adaptation. Understanding the fungal response to host niche is of importance to uncover novel targets for potential symbionts, anti-fungal agents and biotechnical applications.
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http://dx.doi.org/10.1016/j.jprot.2017.03.009DOI Listing
October 2017

Molecular speciation and tissue compartmentation of zinc in durum wheat grains with contrasting nutritional status.

New Phytol 2016 09 9;211(4):1255-65. Epub 2016 May 9.

Plant and Soil Science Section, Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, Frederiksberg C, DK-1871, Denmark.

Low concentration of zinc (Zn) in the endosperm of cereals is a major factor contributing to Zn deficiency in human populations. We have investigated how combined Zn and nitrogen (N) fertilization affects the speciation and localization of Zn in durum wheat (Triticum durum). Zn-binding proteins were analysed with liquid chromatography ICP-MS and Orbitrap MS(2) , respectively. Laser ablation ICP-MS with simultaneous Zn, sulphur (S) and phosphorus (P) detection was used for bioimaging of Zn and its potential ligands. Increasing the Zn and N supply had a major impact on the Zn concentration in the endosperm, reaching concentrations higher than current breeding targets. The S concentration also increased, but S was only partly co-localized with Zn. The mutual Zn and S enrichment was reflected in substantially more Zn bound to small cysteine-rich proteins (apparent size 10-30 kDa), whereas the response of larger proteins (apparent size > 50 kDa) was only modest. Most of the Zn-responsive proteins were associated with redox- and stress-related processes. This study offers a methodological platform to deepen the understanding of processes behind endosperm Zn enrichment. Novel information is provided on how the localization and speciation of Zn is modified during Zn biofortification of grains.
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http://dx.doi.org/10.1111/nph.13989DOI Listing
September 2016

Exploring the Plant-Microbe Interface by Profiling the Surface-Associated Proteins of Barley Grains.

J Proteome Res 2016 Apr 14;15(4):1151-67. Epub 2016 Mar 14.

Agricultural and Environmental Proteomics, Department of Systems Biology, Technical University of Denmark , Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby, Denmark.

Cereal grains are colonized by a microbial community that actively interacts with the plant via secretion of various enzymes, hormones, and metabolites. Microorganisms decompose plant tissues by a collection of depolymerizing enzymes, including β-1,4-xylanases, that are in turn inhibited by plant xylanase inhibitors. To gain insight into the importance of the microbial consortia and their interaction with barley grains, we used a combined gel-based (2-DE coupled to MALDI-TOF-TOF MS) and gel-free (LC-MS/MS) proteomics approach complemented with enzyme activity assays to profile the surface-associated proteins and xylanolytic activities of two barley cultivars. The surface-associated proteome was dominated by plant proteins with roles in defense and stress-responses, while the relatively less abundant microbial (bacterial and fungal) proteins were involved in cell-wall and polysaccharide degradation and included xylanases. The surface-associated proteomes showed elevated xylanolytic activity and contained several xylanases. Integration of proteomics with enzyme assays is a powerful tool for analysis and characterization of the interaction between microbial consortia and plants in their natural environment.
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http://dx.doi.org/10.1021/acs.jproteome.5b01042DOI Listing
April 2016

Monthly oral methylprednisolone pulse treatment in progressive multiple sclerosis.

Mult Scler 2016 06 2;22(7):926-34. Epub 2015 Oct 2.

Danish Multiple Sclerosis Center, Department of Neurology, Rigshospitalet, University of Copenhagen, Denmark.

Background: There is a large unmet need for treatments for patients with progressive multiple sclerosis (MS). Phase 2 studies with cerebrospinal fluid (CSF) biomarker outcomes may be well suited for the initial evaluation of efficacious treatments.

Objective: To evaluate the effect of monthly oral methylprednisolone pulse treatment on intrathecal inflammation in progressive MS.

Methods: In this open-label phase 2A study, 15 primary progressive and 15 secondary progressive MS patients received oral methylprednisolone pulse treatment for 60 weeks. Primary outcome was changes in CSF concentrations of osteopontin. Secondary outcomes were other CSF biomarkers of inflammation, axonal damage and demyelination; clinical scores; magnetic resonance imaging measures of disease activity, magnetization transfer ratio (MTR) and diffusion tensor imaging (DTI); motor evoked potentials; and bone density scans.

Results: We found no change in the CSF concentration of osteopontin, but we observed significant improvement in clinical scores, MTR, DTI and some secondary CSF outcome measures. Adverse events were well-known side effects to methylprednisolone.

Conclusion: Monthly methylprednisolone pulse treatment was safe, but had no effect on the primary outcome. However, improvements in secondary clinical and MRI outcome measures suggest that this treatment regimen may have a beneficial effect in progressive MS.
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http://dx.doi.org/10.1177/1352458515605908DOI Listing
June 2016

Increased probability of repetitive spinal motoneuron activation by transcranial magnetic stimulation after muscle fatigue in healthy subjects.

J Appl Physiol (1985) 2012 Mar 15;112(5):832-40. Epub 2011 Dec 15.

Department of Clinical Neurophysiology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark.

Triple stimulation technique (TST) has previously shown that transcranial magnetic stimulation (TMS) fails to activate a proportion of spinal motoneurons (MNs) during motor fatigue. The depression in size of the TST response, but no attenuation of the conventional motor-evoked potential, suggested increased probability of repetitive spinal MN activation during exercise, even if some MNs failed to discharge by the brain stimulus. Here we used a modified TST [quadruple stimulation (QuadS) and quintuple stimulation (QuintS)] to examine the influence of fatiguing exercise on second and third MN discharges after a single TMS in healthy subjects. This method allows an estimation of the percentage of double and triple discharging MNs. Following a sustained contraction of the abductor digiti minimi muscle at 50% maximal force maintained to exhaustion, the size of QuadS and QuintS responses increased markedly, reflecting that a greater proportion of spinal MNs was activated two or three times by the transcranial stimulus. The size of QuadS responses did not return to precontraction levels during 10-min observation time, indicating long-lasting increase in excitatory input to spinal MNs. In addition, the postexercise behavior of QuadS responses was related to the duration of the contraction, pointing to a correlation between repeated activation of MNs and the subject's ability to maintain force. In conclusion, the study confirmed that an increased fraction of spinal MNs fire more than once in response to TMS when the muscle is fatigued. Repetitive MN firing may provide an adaptive mechanism to maintain motor unit activation and task performance during sustained voluntary activity.
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http://dx.doi.org/10.1152/japplphysiol.00917.2009DOI Listing
March 2012

Responses of barley root and shoot proteomes to long-term nitrogen deficiency, short-term nitrogen starvation and ammonium.

Plant Cell Environ 2011 Dec 16;34(12):2024-37. Epub 2011 Aug 16.

Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Building 224, Søltofts Plads, DK-2800 Kgs. Lyngby, Denmark.

Cereals are major crops worldwide, and improvement of their nitrogen use efficiency is a crucial challenge. In this study proteins responding to N supply in barley roots and shoots were analysed using a proteomics approach, to provide insight into mechanisms of N uptake and assimilation. Control plants grown hydroponically for 33 d with 5 mm nitrate, plants grown under N deficiency (0.5 mm nitrate, 33 d) or short-term N starvation (28 d with 5 mm nitrate followed by 5 d with no N source) were compared. N deficiency caused changes in C and N metabolism and ascorbate-glutathione cycle enzymes in shoots and roots. N starvation altered proteins of amino acid metabolism in roots. Both treatments caused proteome changes in roots that could affect growth. Shoots of plants grown with ammonium as N source (28 d with 5 mm nitrate followed by 5 d with 5 mm ammonium) showed responses similar to N deficient shoots, characterized by turnover of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and increases in proteins of the chloroplastic transcription and translation machinery. Identified proteins in 67 and 49 varying spots in roots and shoots respectively, corresponded to 62 functions and over 80 gene products, considerably advancing knowledge of N responses in barley.
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http://dx.doi.org/10.1111/j.1365-3040.2011.02396.xDOI Listing
December 2011

Proteomes of the barley aleurone layer: A model system for plant signalling and protein secretion.

Proteomics 2011 May 23;11(9):1595-605. Epub 2011 Mar 23.

Enzyme and Protein Chemistry, Department of Systems Biology, Technical University of Denmark, Lyngby, Denmark.

The cereal aleurone layer is of major importance due to its nutritional properties as well as its central role in seed germination and industrial malting. Cereal seed germination involves mobilisation of storage reserves in the starchy endosperm to support seedling growth. In response to gibberellic acid produced by the embryo, the aleurone layer synthesises hydrolases that are secreted to the endosperm for the degradation of storage products. The barley aleurone layer can be separated from the other seed tissues and maintained in culture, allowing the study of the effect of added signalling molecules in an isolated system. These properties have led to its use as a model system for the study of plant signalling and germination. More recently, proteome analysis of the aleurone layer has provided new insight into this unique tissue including identification of plasma membrane proteins and targeted analysis of germination-related changes and the thioredoxin system. Here, analysis of intracellular and secreted proteomes reveals features of the aleurone layer system that makes it promising for investigations of plant protein secretion mechanisms.
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http://dx.doi.org/10.1002/pmic.201000656DOI Listing
May 2011

A recruited protease is involved in catabolism of pyrimidines.

J Mol Biol 2008 May 7;379(2):243-50. Epub 2008 Apr 7.

Department of Cell and Organism Biology, Lund University, SE-22362 Lund, Sweden.

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.
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http://dx.doi.org/10.1016/j.jmb.2008.03.073DOI Listing
May 2008

The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly.

J Mol Biol 2008 Apr 13;377(5):1544-59. Epub 2008 Feb 13.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.

Beta-alanine synthase (betaAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-beta-alanine and N-carbamyl-beta-aminoisobutyrate to the corresponding beta-amino acids. betaASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote betaAS subfamily, we determined the crystal structure of Drosophila melanogaster betaAS to 2.8 A resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central beta-sandwich with a layer of alpha-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster betaAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.
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http://dx.doi.org/10.1016/j.jmb.2008.02.011DOI Listing
April 2008

Crystal structures of yeast beta-alanine synthase complexes reveal the mode of substrate binding and large scale domain closure movements.

J Biol Chem 2007 Dec 4;282(49):36037-47. Epub 2007 Oct 4.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden.

Beta-alanine synthase is the final enzyme of the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of uracil and thymine in higher organisms. The fold of the homodimeric enzyme from the yeast Saccharomyces kluyveri identifies it as a member of the AcyI/M20 family of metallopeptidases. Its subunit consists of a catalytic domain harboring a di-zinc center and a smaller dimerization domain. The present site-directed mutagenesis studies identify Glu(159) and Arg(322) as crucial for catalysis and His(262) and His(397) as functionally important but not essential. We determined the crystal structures of wild-type beta-alanine synthase in complex with the reaction product beta-alanine, and of the mutant E159A with the substrate N-carbamyl-beta-alanine, revealing the closed state of a dimeric AcyI/M20 metallopeptidase-like enzyme. Subunit closure is achieved by a approximately 30 degrees rigid body domain rotation, which completes the active site by integration of substrate binding residues that belong to the dimerization domain of the same or the partner subunit. Substrate binding is achieved via a salt bridge, a number of hydrogen bonds, and coordination to one of the zinc ions of the di-metal center.
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http://dx.doi.org/10.1074/jbc.M705517200DOI Listing
December 2007

Crystallization and preliminary X-ray data analysis of beta-alanine synthase from Drosophila melanogaster.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2007 Oct 19;63(Pt 10):874-7. Epub 2007 Sep 19.

Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.

Beta-alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 A at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 A, beta = 125.8 degrees) and contain 8-10 molecules per asymmetric unit.
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http://dx.doi.org/10.1107/S1744309107042984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2339735PMC
October 2007

A gene duplication led to specialized gamma-aminobutyrate and beta-alanine aminotransferase in yeast.

FEBS J 2007 Apr 12;274(7):1804-17. Epub 2007 Mar 12.

BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark.

In humans, beta-alanine (BAL) and the neurotransmitter gamma-aminobutyrate (GABA) are transaminated by a single aminotransferase enzyme. Apparently, yeast originally also had a single enzyme, but the corresponding gene was duplicated in the Saccharomyces kluyveri lineage. SkUGA1 encodes a homologue of Saccharomyces cerevisiae GABA aminotransferase, and SkPYD4 encodes an enzyme involved in both BAL and GABA transamination. SkPYD4 and SkUGA1 as well as S. cerevisiae UGA1 and Schizosaccharomyces pombe UGA1 were subcloned, over-expressed and purified. One discontinuous and two continuous coupled assays were used to characterize the substrate specificity and kinetic parameters of the four enzymes. It was found that the cofactor pyridoxal 5'-phosphate is needed for enzymatic activity and alpha-ketoglutarate, and not pyruvate, as the amino group acceptor. SkPyd4p preferentially uses BAL as the amino group donor (V(max)/K(m)=0.78 U x mg(-1) x mm(-1)), but can also use GABA (V(max)/K(m)=0.42 U x mg(-1) x mm(-1)), while SkUga1p only uses GABA (V(max)/K(m)=4.01 U x mg(-1) x mm(-1)). SpUga1p and ScUga1p transaminate only GABA and not BAL. While mammals degrade BAL and GABA with only one enzyme, but in different tissues, S. kluyveri and related yeasts have two different genes/enzymes to apparently 'distinguish' between the two reactions in a single cell. It is likely that upon duplication approximately 200 million years ago, a specialized Uga1p evolved into a 'novel' transaminase enzyme with broader substrate specificity.
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http://dx.doi.org/10.1111/j.1742-4658.2007.05729.xDOI Listing
April 2007

The crystal structures of dihydropyrimidinases reaffirm the close relationship between cyclic amidohydrolases and explain their substrate specificity.

J Biol Chem 2006 May 3;281(19):13762-13776. Epub 2006 Mar 3.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden. Electronic address:

In eukaryotes, dihydropyrimidinase catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Here we describe the three-dimensional structures of dihydropyrimidinase from two eukaryotes, the yeast Saccharomyces kluyveri and the slime mold Dictyostelium discoideum, determined and refined to 2.4 and 2.05 angstroms, respectively. Both enzymes have a (beta/alpha)8-barrel structural core embedding the catalytic di-zinc center, which is accompanied by a smaller beta-sandwich domain. Despite loop-forming insertions in the sequence of the yeast enzyme, the overall structures and architectures of the active sites of the dihydropyrimidinases are strikingly similar to each other, as well as to those of hydantoinases, dihydroorotases, and other members of the amidohydrolase superfamily of enzymes. However, formation of the physiologically relevant tetramer shows subtle but nonetheless significant differences. The extension of one of the sheets of the beta-sandwich domain across a subunit-subunit interface in yeast dihydropyrimidinase underlines its closer evolutionary relationship to hydantoinases, whereas the slime mold enzyme shows higher similarity to the noncatalytic collapsin-response mediator proteins involved in neuron development. Catalysis is expected to follow a dihydroorotase-like mechanism but in the opposite direction and with a different substrate. Complexes with dihydrouracil and N-carbamyl-beta-alanine obtained for the yeast dihydropyrimidinase reveal the mode of substrate and product binding and allow conclusions about what determines substrate specificity, stereoselectivity, and the reaction direction among cyclic amidohydrolases.
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http://dx.doi.org/10.1074/jbc.M513266200DOI Listing
May 2006

Purification, crystallization and X-ray diffraction analysis of dihydropyrimidinase from Dictyostelium discoideum.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2006 Jan 16;62(Pt 1):36-8. Epub 2005 Dec 16.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

Dihydropyrimidinase (EC 3.5.2.2) is the second enzyme in the reductive pyrimidine-degradation pathway and catalyses the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. The recombinant enzyme from the slime mould Dictyostelium discoideum was overexpressed, purified and crystallized by the vapour-diffusion method. One crystal diffracted to better than 1.8 A resolution on a synchrotron source and was shown to belong to space group I222, with unit-cell parameters a = 84.6, b = 89.6, c = 134.9 A and one molecule in the asymmetric unit.
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http://dx.doi.org/10.1107/S174430910503976XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2150923PMC
January 2006

Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2005 Apr 12;61(Pt 4):359-62. Epub 2005 Mar 12.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.
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http://dx.doi.org/10.1107/S174430910500610XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1952421PMC
April 2005

Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri.

Acta Crystallogr D Biol Crystallogr 2003 Jul 27;59(Pt 7):1267-9. Epub 2003 Jun 27.

Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.

In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.
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http://dx.doi.org/10.1107/s0907444903009120DOI Listing
July 2003

Failure of activation of spinal motoneurones after muscle fatigue in healthy subjects studied by transcranial magnetic stimulation.

J Physiol 2003 Aug 24;551(Pt 1):345-56. Epub 2003 Jun 24.

Department of Clinical Neurophysiology 3063, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, DK-2100 Copenhagen, Denmark.

During a sustained maximal effort a progressive decline in the ability to drive motoneurones (MNs) develops. We used the recently developed triple stimulation technique (TST) to study corticospinal conduction after fatiguing exercise in healthy subjects. This method employs a collision technique to estimate the proportion of motor units activated by a transcranial magnetic stimulus. Following a sustained contraction of the abductor digiti minimi muscle at 50 % maximal force maintained to exhaustion there was an immediate reduction of the TST response from > 95 % to about 60 %. This effect recovered to control levels within 1 min and implies that a decreased number of spinal MNs were excited. Additional TST experiments after maximal and submaximal efforts showed that the decrease in size of the TST response was related to duration and strength of exercise. Motor evoked potentials (MEPs) after conventional transcranial magnetic stimulation (TMS) and responses to peripheral nerve stimulation were recorded following the same fatigue protocol. The size of both the MEPs and the peripheral responses increased after the contraction and were in direct contrast to the decrease in size of the TST response. This points to increased probability of repetitive spinal MN activation during fatigue even if some MNs in the pool failed to discharge. Silent period duration following cortical stimulation lengthened by an average of 55 ms after the contraction and recovered within a time course similar to that of the TST response depression. Overall, the results suggest that the outflow from the motor cortex could become insufficient to drive all spinal MNs to discharge when the muscle is fatigued and that complex interactions between failure of activation and compensatory mechanisms to maintain motor unit activation occur during sustained voluntary activity. When inability to maintain force occurs during submaximal effort, failure of activation of motor units is predominant.
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http://dx.doi.org/10.1113/jphysiol.2003.043562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2343163PMC
August 2003

Dihydropyrimidine amidohydrolases and dihydroorotases share the same origin and several enzymatic properties.

Nucleic Acids Res 2003 Mar;31(6):1683-92

Eukaryote Molecular Biology, BioCentrum-DTU, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark.

Slime mold, plant and insect dihydropyrimidine amidohydrolases (DHPases, EC 3.5.2.2), which catalyze the second step of pyrimidine and several anti-cancer drug degradations, were cloned and shown to functionally replace a defective DHPase enzyme in the yeast Saccharomyces kluyveri. The yeast and slime mold DHPases were over-expressed, shown to contain two zinc ions, characterized for their properties and compared to those of the calf liver enzyme. In general, the kinetic parameters varied widely among the enzymes, the mammalian DHPase having the highest catalytic efficiency. The ring opening was catalyzed most efficiently at pH 8.0 and competitively inhibited by the reaction product, N-carbamyl-beta-alanine. At lower pH values DHPases catalyzed the reverse reaction, the closing of the ring. Apparently, eukaryote DHPases are enzymatically as well as phylogenetically related to the de novo biosynthetic dihydroorotase (DHOase) enzymes. Modeling studies showed that the position of the catalytically critical amino acid residues of bacterial DHOases and eukaryote DHPases overlap. Therefore, only a few modifications might have been necessary during evolution to convert the unspecialized enzyme into anabolic and catabolic ones.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC152861PMC
http://dx.doi.org/10.1093/nar/gkg258DOI Listing
March 2003
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