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Søren Bak

Publications and source records attributed to Søren Bak.

At least 19 recordsLinked to original sources
PubMed2005

Determination of catalytic key amino acids and UDP sugar donor specificity of the cyanohydrin glycosyltransferase UGT85B1 from Sorghum bicolor. Molecular modeling substantiated by site-specific mutagenesis and biochemical analyses.

Plants produce a plethora of structurally diverse natural products. The final step in their biosynthesis is often a glycosylation step catalyzed by a family 1 glycosyltransferase (GT). In biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor, the UDP-glucosyltransferase UGT85B1 catalyzes the conversion of p-hydroxymandelonitrile into dhurrin. A structural model of UGT85B1 was built based on hydrophobic cluster analysis and the crystal structures of two bacterial GTs, GtfA and GtfB, which each showed approximately 15% overall amino acid sequence identity to UGT85B1. The model enabled predictions about amino acid residues important for catalysis and sugar donor specificity. p-Hydroxymandelonitrile and UDP-glucose (Glc) were predicted to be positioned within hydrogen-bonding distance to a glutamic acid residue in position 410 facilitating sugar transfer. The acceptor was packed within van der Waals distance to histidine H23. Serine S391 and arginine R201 form hydrogen bonds to the pyrophosphate part of UDP-Glc and hence stabilize binding of the sugar donor. Docking of UDP sugars predicted that UDP-Glc would serve as the sole donor sugar in UGT85B1. This was substantiated by biochemical analyses. The predictive power of the model was validated by site-directed mutagenesis of selected residues and using enzyme assays. The modeling approach has provided a tool to design GTs with new desired substrate specificities for use in biotechnological applications. The modeling identified a hypervariable loop (amino acid residues 156-188) that contained a hydrophobic patch. The involvement of this loop in mediating binding of UGT85B1 to cytochromes P450, CYP79A1, and CYP71E1 within a dhurrin metabolon is discussed.

Amino Acid Sequence↗
PubMed2005

Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers. Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology.

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.

Biological Transport, Active↗
PubMed2005

Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome.

Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.

Amino Acids↗
PubMed2005

Metabolon formation and metabolic channeling in the biosynthesis of plant natural products.

Metabolon formation and metabolic channeling in plant secondary metabolism enable plants to effectively synthesize specific natural products and to avoid metabolic interference. Channeling can involve different cell types, take advantage of compartmentalization within the same cell or proceed directly within a metabolon. New experimental approaches document the importance of channeling in the synthesis of isoprenoids, alkaloids, phenylpropanoids, flavonoids and cyanogenic glucosides. Metabolon formation and metabolic channeling in natural-product synthesis facilitate attempts to genetically engineer new pathways into plants to improve their content of valuable natural products. They also offer the opportunity to introduce new traits by genetic engineering to produce plant cultivars that adhere to the principle of substantial equivalence.

Alkaloids↗
PubMed2004

Spot shape modelling and data transformations for microarrays.

MOTIVATION: To study lowly expressed genes in microarray experiments, it is useful to increase the photometric gain in the scanning. However, a large gain may cause some pixels for highly expressed genes to become saturated. Spatial statistical models that model spot shapes on the pixel level may be used to infer information about the saturated pixel intensities. Other possible applications for spot shape models include data quality control and accurate determination of spot centres and spot diameters. RESULTS: Spatial statistical models for spotted microarrays are studied including pixel level transformations and spot shape models. The models are applied to a dataset from 50mer oligonucleotide microarrays with 452 selected Arabidopsis genes. Logarithmic, Box-Cox and inverse hyperbolic sine transformations are compared in combination with four spot shape models: a cylindric plateau shape, an isotropic Gaussian distribution and a difference of two-scaled Gaussian distribution suggested in the literature, as well as a proposed new polynomial-hyperbolic spot shape model. A substantial improvement is obtained for the dataset studied by the polynomial-hyperbolic spot shape model in combination with the Box-Cox transformation. The spatial statistical models are used to correct spot measurements with saturation by extrapolating the censored data. AVAILABILITY: Source code for R is available at http://www.matfys.kvl.dk/~ekstrom/spotshapes/

Algorithms↗
PubMed2004

Cyanogenic glucosides and plant-insect interactions.

Cyanogenic glucosides are phytoanticipins known to be present in more than 2500 plant species. They are considered to have an important role in plant defense against herbivores due to bitter taste and release of toxic hydrogen cyanide upon tissue disruption. Some specialized herbivores, especially insects, preferentially feed on cyanogenic plants. Such herbivores have acquired the ability to metabolize cyanogenic glucosides or to sequester them for use in their predator defense. A few species of Arthropoda (within Diplopoda, Chilopoda, Insecta) are able to de novo synthesize cyanogenic glucosides and, in addition, some of these species are able to sequester cyanogenic glucosides from their host plant (Zygaenidae). Evolutionary aspects of these unique plant-insect interactions with focus on the enzyme systems involved in synthesis and degradation of cyanogenic glucosides are discussed.

Animals↗
PubMed2004

Biosynthesis of the nitrile glucosides rhodiocyanoside A and D and the cyanogenic glucosides lotaustralin and linamarin in Lotus japonicus.

Lotus japonicus was shown to contain the two nitrile glucosides rhodiocyanoside A and rhodiocyanoside D as well as the cyanogenic glucosides linamarin and lotaustralin. The content of cyanogenic and nitrile glucosides in L. japonicus depends on plant developmental stage and tissue. The cyanide potential is highest in young seedlings and in apical leaves of mature plants. Roots and seeds are acyanogenic. Biosynthetic studies using radioisotopes demonstrated that lotaustralin, rhodiocyanoside A, and rhodiocyanoside D are derived from the amino acid l-Ile, whereas linamarin is derived from Val. In silico homology searches identified two cytochromes P450 designated CYP79D3 and CYP79D4 in L. japonicus. The two cytochromes P450 are 94% identical at the amino acid level and both catalyze the conversion of Val and Ile to the corresponding aldoximes in biosynthesis of cyanogenic glucosides and nitrile glucosides in L. japonicus. CYP79D3 and CYP79D4 are differentially expressed. CYP79D3 is exclusively expressed in aerial parts and CYP79D4 in roots. Recombinantly expressed CYP79D3 and CYP79D4 in yeast cells showed higher catalytic efficiency with l-Ile as substrate than with l-Val, in agreement with lotaustralin and rhodiocyanoside A and D being the major cyanogenic and nitrile glucosides in L. japonicus. Ectopic expression of CYP79D2 from cassava (Manihot esculenta Crantz.) in L. japonicus resulted in a 5- to 20-fold increase of linamarin content, whereas the relative amounts of lotaustralin and rhodiocyanoside A/D were unaltered.

Cytochrome P-450 Enzyme System↗
PubMed2004

Comparative genomics of rice and Arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a dicot.

Data mining methods have been used to identify 356 Cyt P450 genes and 99 related pseudogenes in the rice (Oryza sativa) genome using sequence information available from both the indica and japonica strains. Because neither of these genomes is completely available, some genes have been identified in only one strain, and 28 genes remain incomplete. Comparison of these rice genes with the 246 P450 genes and 26 pseudogenes in the Arabidopsis genome has indicated that most of the known plant P450 families existed before the monocot-dicot divergence that occurred approximately 200 million years ago. Comparative analysis of P450s in the Pinus expressed sequence tag collections has identified P450 families that predated the separation of gymnosperms and flowering plants. Complete mapping of all available plant P450s onto the Deep Green consensus plant phylogeny highlights certain lineage-specific families maintained (CYP80 in Ranunculales) and lineage-specific families lost (CYP92 in Arabidopsis) in the course of evolution.

Arabidopsis↗
PubMed2003

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime.

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.

Amino Acid Sequence↗
PubMed2003

On the origin of family 1 plant glycosyltransferases.

The phylogeny of highly divergent multigene families is often difficult to validate but can be substantiated by inclusion of data outside of the phylogeny, such as signature motifs, intron splice site conservation, unique substitutions of conserved residues, similar gene functions, and out groups. The Family 1 Glycosyltransferases (UGTs) comprises such a highly divergent, polyphyletic multigene family. Phylogenetic comparisons of UGTs from plants, animals, fungi, bacteria, and viruses reveal that plant UGTs represent three distinct clades. The majority of the plant sequences appears to be monophyletic and have diverged after the bifurcation of the animal/fungi/plant kingdoms. The two minor clades contain the sterol and lipid glycosyltransferases and each show more homology to non-plant sequences. The lipid glycosyltransferase clade is homologous to bacterial lipid glycosyltransferases and reflects the bacterial origin of chloroplasts. The fully sequenced Arabidopsis thaliana genome contains 120 UGTs including 8 apparent pseudogenes. The phylogeny of plant glycosyltransferases is substantiated with complete phylogenetic analysis of the A. thaliana UGT multigene family, including intron-exon organization and chromosomal localization.

Amino Acid Sequence↗
PubMed2003

The in vitro substrate regiospecificity of recombinant UGT85B1, the cyanohydrin glucosyltransferase from Sorghum bicolor.

The in vitro substrate specificity of UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase from Sorghum bicolor (UGT85B1) was examined using a range of potential acceptor molecules, including cyanohydrins, terpenoids, phenolics, hexanol derivatives and plant hormones. Qualitative enzyme activity assays employing 20 different putative substrates were performed and 15 proved to be glucosylated using recombinant UGT85B1 isolated from Escherichia coli. K(m) and k(cat) values were determined for nine of these substrates including mandelonitrile, geraniol, nerol and beta-citronellol, 2-hydroxy-3-methoxybenzyl alcohol, 1-hexanol, cis-3-hexen-1-ol, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol. UGT85B1 has a rather broad substrate specificity in vitro but shows regiospecificity, demanding the presence of a sterically unhindered hydroxyl group e.g. as part of a cyanohydrin function, as a primary alcohol or as a phenolic hydroxyl group and being influenced by the stereochemistry and/or interactive chemistry of the substituents on the hydroxyl-bearing carbon atom.

Arabidopsis↗
PubMed2003

Pain phenomena and possible mechanisms in patients with painful polyneuropathy.

Painful polyneuropathy is a common neuropathic pain condition characterized by different typical pain phenomena and symptoms. The present study determined the frequency of pain phenomena and signs in painful polyneuropathy, and compared the symptomatology in patients with signs of increased small fiber response with that in patients with signs of deafferentation. Eighty-one consecutive patients with painful polyneuropathy were studied. The most common pain phenomena were deep aching pain (88%) and pain on pressure (69%), followed by pain paroxysms (59%) and less frequently pain on light touch (31%). Patients with increased cold and heat detection thresholds were more likely to have pain paroxysms (odds ratio (OR) 2.5 and 5.2) and patients with pain summation on repetitive mechanical stimulation more often had touch-evoked pain (OR=4.0) than patients without these phenomena. Findings compatible with increased small fiber response were found in six patients (7.4%), in 41 (50.6%) unequivocal signs of deafferentation were found, and 34 patients (42%) could not be classified. There was no significant difference in presenting symptoms between these groups. In conclusion, in painful polyneuropathy, (1). deep aching pain is the most frequently reported pain symptom; (2). the association between pain paroxysms with decreased small fiber function and touch-evoked pain with abnormal pain summation on mechanical stimulation indicate that central nervous system mechanisms are responsible for these symptoms; (3). sensitized small fibers as the single mechanism of pain is rare; and (4). pain symptomatology cannot predict pain mechanisms as being mainly deafferentation or sensitized small fibers.

Adult↗
PubMed2003

Plant cytochromes P450: tools for pharmacology, plant protection and phytoremediation.

Cytochromes P450 catalyse extremely diverse and often complex regiospecific and/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important functions in plant defence and metabolic engineering of P450s is a prime target to improve plant defence against insects and pathogens. Herbicides, pollutants and other xenobiotics are metabolised by some plant P450 enzymes. These P450s are tools to modify herbicide tolerance, as selectable markers and for bioremediation.

Antineoplastic Agents↗
PubMed2003

CYP83A1 and CYP83B1, two nonredundant cytochrome P450 enzymes metabolizing oximes in the biosynthesis of glucosinolates in Arabidopsis.

In the glucosinolate pathway, the postoxime enzymes have been proposed to have low specificity for the side chain and high specificity for the functional group. Here, we provide biochemical evidence for the functional role of the two cytochromes P450, CYP83A1 and CYP83B1, from Arabidopsis in oxime metabolism in the biosynthesis of glucosinolates. In a detailed analysis of the substrate specificities of the recombinant enzymes heterologously expressed in yeast (Saccharomyces cerevisiae), we show that aliphatic oximes derived from chain-elongated homologs of methionine are efficiently metabolized by CYP83A1, whereas CYP83B1 metabolizes these substrates with very low efficiency. Aromatic oximes derived from phenylalanine, tryptophan, and tyrosine are metabolized by both enzymes, although CYP83B1 has higher affinity for these substrates than CYP83A1, particularly in the case of indole-3-acetaldoxime, where there is a 50-fold difference in K(m) value. The data show that CYP83A1 and CYP83B1 are nonredundant enzymes under physiologically normal conditions in the plant. The ability of CYP83A1 to metabolize aromatic oximes, albeit at small levels, explains the presence of indole glucosinolates at various levels in different developmental stages of the CYP83B1 knockout mutant, rnt1-1. Plants overexpressing CYP83B1 contain elevated levels of aliphatic glucosinolates derived from methionine homologs, whereas the level of indole glucosinolates is almost constant in the overexpressing lines. Together with the previous characterization of the members of the CYP79 family involved in oxime production, this work provides a framework for metabolic engineering of glucosinolates and for further dissection of the glucosinolate pathway.

Arabidopsis↗
PubMed2003

Stroke research in GenomEUtwin.

Stroke is one of the leading causes of severe disability and death in the world. In the present article we outline possibilities and limitations for future stroke research within the GenomEUtwin. The combined sample of twins born before 1958 from Denmark, Finland, and Sweden, and available for follow-up into the second millennium for non-fatal and fatal stroke events through national inpatient and death registers exceeds 70,000 twin pairs. This sample size will enable the study of genetic influences on stroke and major stroke subtypes. Large samples of twins in GenomEUtwin have been followed up repeatedly through interviews and questionnaires concerning a variety of exposures and potential risk factors for stroke. We briefly outline how this information can be combined with the health register information for epidemiologic and genetic epidemiologic studies of stroke. We also present the number of twin pairs concordant and discordant for stroke in Denmark, Finland and Sweden, and time lags between events for twins concordant for stroke. This information illustrates that the number of affected sib pairs for linkage studies is relatively limited, but the sample sizes are promising for association studies.

Denmark↗
PubMed2003

Risk of stroke associated with nonsteroidal anti-inflammatory drugs: a nested case-control study.

BACKGROUND AND PURPOSE: Nonsteroidal anti-inflammatory drugs (NSAIDs) have been associated with bleeding complications and may affect the risk of hemorrhagic stroke through inhibition of platelet cyclooxygenase-1. We performed a population-based case-control study to estimate the risk of intracerebral hemorrhage, subarachnoid hemorrhage, and ischemic stroke in users of NSAIDs. METHODS: We used a population-based patient registry to identify all patients with a first-ever stroke discharge diagnosis in the period of 1994 to 1999. All diagnoses were validated according to predefined criteria. We selected 40 000 random controls from the background population. Information on drug use for cases and controls was retrieved from a prescription registry. Odds ratios were adjusted for age, sex, calendar year, and use of other medication. To evaluate the effect of various potential confounders not recorded in the register, we performed separate analyses on data from 2 large population-based surveys with more detailed information on risk factors. RESULTS: The cases were classified as intracerebral hemorrhage (n=659), subarachnoid hemorrhage (n=208), and ischemic stroke (n=2717). The adjusted odds ratio of stroke in current NSAID users compared with never users was 1.2 (95% CI, 0.9 to 1.6) for intracerebral hemorrhage, 1.2 (95% CI, 0.7 to 2.1) for subarachnoid hemorrhage and 1.2 (95% confidence interval, 1.0 to 1.4) for ischemic stroke. The survey data indicated that additional confounder control would not have led to an increase in relative risk estimates. CONCLUSIONS: Current exposure to NSAIDs is not a risk factor for intracerebral hemorrhage or subarachnoid hemorrhage. Furthermore, NSAIDs probably offer no protection against first-ever ischemic stroke.

Adult↗
PubMed2002

Selective serotonin reuptake inhibitors and the risk of stroke: a population-based case-control study.

BACKGROUND AND PURPOSE: Selective serotonin reuptake inhibitors (SSRIs) have been associated with increased risk of bleeding complications, possibly as a result of inhibition of platelet aggregation. Little is known about the risk of intracerebral hemorrhage in users of SSRIs and whether the effect on platelet aggregation reduces the risk of ischemic stroke. We used population-based data to estimate the risk of hemorrhagic and ischemic stroke in users of SSRIs. METHODS: We performed a nested case-control study in Funen County (465 000 inhabitants), Denmark. All patients with a first-ever stroke discharge diagnosis in the period of 1994 to 1999 were identified, and a validated diagnosis of stroke was reached in 4765 cases. In all, 40 000 controls were randomly selected from the background population. Information on drug use for cases and controls was retrieved from a prescription registry with full coverage of the county. Odds ratios were adjusted for age, sex, calendar year, and use of other medication. To evaluate the effect of various potential confounders not recorded in the register data, we performed separate analyses on data from 2 large population-based surveys with more detailed information on risk factors. RESULTS: Of 659 patients with hemorrhagic stroke, 21 were current users of SSRIs. The adjusted odds ratio of hemorrhagic stroke in current SSRI users compared with never users was 1.0 [95% confidence interval (CI), 0.6 to 1.6]. Of 2717 patients with ischemic stroke, 100 were current users of SSRIs, and the adjusted odds ratio of ischemic stroke in cases compared with controls was 1.1 (95% CI, 0.9 to 1.4). The survey data indicated that additional confounder control would not have led to an increase in the relative risk estimates. CONCLUSIONS: Current exposure to SSRIs is not associated with increased risk of intracerebral hemorrhage and is probably not associated with a decreased risk of ischemic stroke.

Adult↗