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A Bacher

Publications and source records attributed to A Bacher.

At least 73 records · Page 4Linked to original sources
PubMed2000

Biosynthesis of terpenoids: 1-deoxy-D-xylulose-5-phosphate reductoisomerase from Escherichia coli is a class B dehydrogenase.

1-Deoxy-D-xylulose-5-phosphate is converted into 2-C-methyl-D-erythritol-4-phosphate by the catalytic action of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (Dxr protein) using NADPH as cofactor. The stereochemical features of this reaction were investigated in in vitro experiments with the recombinant Dxr protein of Escherichia coli using (4R)- or (4S)-[4-(2)H(1)]NADPH as coenzyme. The enzymatically formed 2-C-methyl-D-erythritol-4-phosphate was isolated and converted into 1,2:3,4-di-O-isopropylidene-2-C-methyl-D-erythritol; NMR spectroscopic investigation of this derivative indicated that only (4S)-[4-(2)H(1)]NADPH affords 2-C-methyl-D-erythritol-4-phosphate labelled exclusively in the H(Re) position of C-1. Stereospecific transfer of H(Si) from C-4 of the cofactor identifies the Dxr protein of E. coli as a class B dehydrogenase.

Aldose-Ketose Isomerases↗
PubMed2000

Biosynthesis of riboflavin in plants. The ribA gene of Arabidopsis thaliana specifies a bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4-phosphate synthase.

A cDNA segment from Arabidopsis thaliana with similarity to the ribA gene of Bacillus subtilis was sequenced. A similar gene was cloned from tomato. The open reading frame of A. thaliana was fused to the malE gene of Escherichia coli and was expressed in a recombinant E. coli strain. The recombinant fusion protein was purified and shown to have GTP cyclohydrolase II activity as well as 3,4-dihydroxy-2-butanone 4-phosphate synthase activity. The cognate gene was amplified by polymerase chain reaction from chromosomal Arabidopsis DNA and was shown to contain six introns. Intron 4 is located in the region connecting the GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the putative domains catalyzing the two reaction steps. By comparison with the bacterial ribA gene, the Arabidopsis gene contains an additional 5' element specifying about 120 amino acid residues. This segment contains numerous serine and threonine residues and does not show similarity with other known sequences. The N-terminal segment is not required for catalytic activity and is likely to serve as signal sequence for import into chloroplasts.

Amino Acid Sequence↗
PubMed2000

Flavin-protein interactions in flavocytochrome b2 as studied by NMR after reconstitution of the enzyme with 13C- and 15N-labelled flavin.

A new procedure was devised for reversibly removing the flavin from flavocytochrome b2. It allowed reconstitution with selectively enriched 13C- and 15N-labelled FMN for an NMR analysis of the chemical shifts of the enriched positions as well as that of 31P. From these measurements, it was possible to deduce information about the hydrogen-bonding pattern of FMN in the protein, the hybridization states of the nitrogen atoms and (in part) the pi-electron distribution. The carbonyl groups at C(2) and C(4) and the nitrogen atoms N(1) and N(5) form hydrogen bonds to the apoenzyme in both redox states. Nevertheless, according to 15N-chemical shifts, the bond from the protein to N(3) is very weak in both redox states, whereas that to N(5) is strong for the oxidized state, and is weakened upon flavin reduction. On the other hand, the 13C-NMR results indicate that the C(2) and C(4) carbonyl oxygens form stronger hydrogen bonds with the enzyme than most other flavoproteins in both redox states. From coupling constant measurements it is shown that the N(3) proton is not solvent accessible. Although no N-H coupling constant could be measured for N(5) in the reduced state due to lack of resolution, N(5) is clearly protonated in flavocytochrome b2 as in all other known flavoproteins. With respect to N(10), it is more sp3-hybridized in the oxidized state than in free FMN, whereas the other nitrogen atoms show a nearly planar structure. In the reduced state, N(5) and N(10) in bound FMN are both more sp3-hybridized than in free FMN, but N(5) exhibits a higher degree of sp3-hybridization than N(10), which is only slightly shifted out of the isoalloxazine plane. In addition, two-electron reduction of the enzyme leads to anion formation on N(1), as indicated by its 15N-chemical shift of N(1) and characteristic upfield shifts of the resonances of C(2), C(4) and C(4a) compared to the oxidized state, as observed for most flavoproteins. 31P-NMR measurements show that the phosphate geometry has changed in enzyme bound FMN compared to the free flavin in water, indicating a strong interaction of the phosphate group with the apoenzyme.

Binding Sites↗
PubMed2000

Supraglottic combined frequency jet ventilation versus subglottic monofrequent jet ventilation in patients undergoing microlaryngeal surgery.

UNLABELLED: We compared the efficacy of gas exchange during supraglottic combined-frequency jet ventilation via a jet ventilation laryngoscope and during monofrequent jet ventilation via the Mon-Jet catheter (Xomed, Jacksonville, FL). Twenty-three anesthetized (propofol, fentanyl, vecuronium) patients undergoing microlaryngeal surgery were prospectively studied and randomly assigned to one of two groups. The patients' lungs were ventilated with combined-frequency jet ventilation (10 min, 15 and 600 breaths/min, inspiration/expiration time ratio = 1, driving pressure 750-1500 mm Hg), monofrequent (low-frequency group: 15 breaths/min; high-frequency group: 600 breaths/min) jet ventilation (20 min), and again combined-frequency jet ventilation (15 min). PaO(2), PaCO(2), and the inspiratory oxygen fraction (FIO(2)) were measured. Wilcoxon's signed rank test was applied. During monofrequent jet ventilation, PaCO(2) increased and the PaO(2)/FIO(2) decreased significantly (P < 0.05) as compared with combined-frequency jet ventilation (low-frequency group: PaCO(2) from 39.4 +/- 3.3 to 50. 8 +/- 8.0 mm Hg, PaO(2)/FIO(2) from 306 +/- 100 to 225 +/- 94 mm Hg; high-frequency group: PaCO(2) from 36.7 +/- 7.2 to 60.3 +/- 6.1 mm Hg, PaO(2)/FIO(2) from 429 +/- 87 to 190 +/- 51 mm Hg; mean +/- SD). After switching back to combined-frequency jet ventilation, PaCO(2) decreased and PaO(2)/FIO(2) increased to baseline levels. We conclude that gas exchange during microlaryngeal surgery can be more easily maintained with supraglottic combined-frequency jet ventilation than with subglottic monofrequent jet ventilation via the Mon-Jet catheter. IMPLICATIONS: This study demonstrates that the combination of high- and low-frequency supraglottic jet ventilation via a jet ventilation laryngoscope provides a better pulmonary gas exchange and allows more accurate airway pressure monitoring during microlaryngeal surgery than subglottic monofrequent jet ventilation via an endotracheal catheter.

Adult↗
PubMed2000

Respiratory efficacy of subglottic low-frequency, subglottic combined-frequency, and supraglottic combined-frequency jet ventilation during microlaryngeal surgery.

UNLABELLED: We tested the respiratory efficacy of different jet ventilation techniques (subglottic low-frequency versus subglottic combined-frequency and subglottic combined-frequency versus supraglottic combined frequency) in patients undergoing microlaryngeal surgery. The PaCO(2) and the quotient of arterial oxygen tension (PaO(2)) over FIO(2) were measured. After anesthetic induction (propofol, remifentanil, vecuronium), an endotracheal Mon-Jet catheter (Xomed, Jacksonville, FL) for subglottic jet ventilation and a laryngoscope for supraglottic jet ventilation (Carl Reiner G.m.b.H., Vienna, Austria) were inserted. In Group 1 (n = 18), subglottic low-frequency (15 breaths/min), combined-frequency (600 and 15 breaths/min), and low-frequency jet ventilation was subsequently performed (15 min each). In Group 2 (n = 19), the sequence was supraglottic, subglottic, and supraglottic combined-frequency jet ventilation. The driving pressures were initially adjusted to achieve normocapnia and were not changed during the entire study period. The FIO(2) was measured endotracheally. The Wilcoxon's signed rank test was applied. In Group 1, PaCO(2) and PaO(2)/FIO(2) improved significantly after switching from subglottic low-frequency to subglottic combined-frequency jet ventilation (PaCO(2), from 46.6 +/-8.3 to 42.1+/-8.1 mm Hg; PaO(2)/FIO(2), from 311+/-144 to 361+/-141 mm Hg; P<0.05). In Group 2, PaCO(2) increased and PaO(2)/FIO(2) decreased significantly after switching from supraglottic to subglottic combined-frequency jet ventilation (PaCO(2), from 39.4+/-7.1 to 45.9+/-7.5 mm Hg; PaO(2)/FIO(2), from 415+/-114 to 351+/-129 mm Hg; P<0.05). We conclude that subglottic combined-frequency jet ventilation is less effective than supraglottic combined-frequency ventilation, but more effective than subglottic low-frequency jet ventilation. IMPLICATIONS: The combination of high and low respiratory frequencies (600 and 15 breaths/min) improves pulmonary gas exchange during subglottic jet ventilation via an endotracheal catheter. However, subglottic combined-frequency jet ventilation is less effective than supraglottic combined-frequency jet ventilation via a jet ventilation laryngoscope.

Adult↗
PubMed2000

Effects of pentobarbital and isoflurane on conditioned learning after transient global cerebral ischemia in rabbits.

BACKGROUND: The acquisition of a conditioned eyeblink response has been used extensively to study the neurologic substrates of learning and memory. We examined the effects of the anesthetics isoflurane and pentobarbital, or hypothermia (30 degrees C), on the ability of rabbits to acquire an eyeblink conditioned response after 6.5 min of cerebral ischemia. METHODS: New Zealand white rabbits (n = 48) were randomly assigned to sham, normothermic, hypothermic, isoflurane, or pentobarbital groups. In the normothermic, hypothermic, isoflurane, and pentobarbital groups, 6.5 min of global cerebral ischemia was produced. In animals randomized to the isoflurane and pentobarbital groups, a pattern of burst suppression was achieved on the electroencephalogram before the start of the ischemic episode. Animals in the hypothermia group were cooled to 30 degrees C before ischemia. Seven days after ischemia, eyeblink training was started using an audible tone presented for 100 ms as the conditioned stimulus. The unconditioned stimulus was an air puff directed at the cornea. The delay between the end of conditioned stimulus and the start of the unconditioned stimulus (the trace interval) was 300 ms in duration. A conditioned response was defined as an eyeblink that was initiated during the trace interval. Eighty trials per day and 15 days of training were delivered. RESULTS: Neurologic deficits were greatest in the normothermia group, and these animals also had fewer conditioned responses than those in the sham, hypothermia, or pentobarbital groups. Animals in the isoflurane group had an intermediate number of conditioned responses that was not significantly different from the normothermia group. CONCLUSIONS: This study demonstrates that a brief episode of cerebral ischemia results in the impairment of associative learning. Hypothermia and burst-suppressive doses of pentobarbital were able to improve neurobehavioral outcome as measured by ability to acquire a trace conditioned response.

Adjuvants, Anesthesia↗
PubMed2000

Auxin biosynthesis in maize kernels.

Auxin biosynthesis was analyzed in a maize (Zea mays) kernel culture system in which the seeds develop under physiological conditions similar to the in vivo situation. This system was modified for precursor feeding experiments. Tryptophan (Trp) is efficiently incorporated into indole-3-acetic acid (IAA) with retention of the 3, 3' bond. Conversion of Trp to IAA is not competed by indole. Labeling with the general precursors [U-(13)C(6)]glucose and [1, 2-(13)C(2)]acetate followed by retrobiosynthetic analysis strongly suggest that Trp-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. The synthesis of IAA from indole glycerol phosphate and IAA formation via condensation of indole with an acetyl-coenzyme A or phosphoenolpyruvate derived metabolite can be excluded.

Amino Acids↗
PubMed2000

Biosynthesis of vitamin b2 (riboflavin).

The biosynthesis of one riboflavin molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate as substrates. The imidazole ring of GTP is hydrolytically opened, yielding a 4, 5-diaminopyrimidine which is converted to 5-amino-6-ribitylamino-2, 4(1H,3H)-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione with 3, 4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate affords 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione, which is recycled in the biosynthetic pathway. The structure of the biosynthetic enzyme, 6,7-dimethyl-8-ribityllumazine synthase, has been studied in considerable detail.

Bacillus subtilis↗
PubMed2000

Three-dimensional computer morphometry of the maxilla and face in infants with Pierre Robin sequence--a comparative study.

OBJECTIVE: To analyze the morphology of the maxillary crest in infants with Pierre Robin sequence using an anthropometric coordinate system and to compare the data with those of healthy infants. SETTING: The study was performed at a craniofacial center servicing a large geographic area. PARTICIPANTS: The study involved eight infants aged 1-28 days (average, 7 days) with an established diagnosis of Pierre Robin sequence and six healthy infants aged 1-43 days (average, 22 days). MAIN OUTCOME MEASURES: Physical models of the maxilla and face obtained by alginate replication were analyzed by computer morphometry yielding the three-dimensional topology of the maxillary crest. RESULTS: The maxillary crest of children with Pierre Robin sequence shows an increased inclination relative to the transverse plane (30 +/- 3.9 degrees) as compared with that of healthy infants (20 +/- 2.9 degrees). The maxillary crest of the patients is shortened in the sagittal direction by comparison with healthy controls. CONCLUSIONS: The increased inclination of the maxilla in infants with Pierre Robin sequence may aggravate the retroposition of the mandible and may thus be a pathogenetic factor contributing to the severe respiratory problems.

Alginates↗
PubMed1999

EPR, ENDOR, and TRIPLE resonance spectroscopy on the neutral flavin radical in Escherichia coli DNA photolyase.

Ultraviolet radiation promotes the formation of a cyclobutane ring between adjacent pyrimidine residues on the same DNA strand to form a pyrimidine dimer. Such dimers may be restored to their monomeric forms through the action of a light-absorbing enzyme named DNA photolyase. The redox-active cofactor involved in the light-induced electron transfer reactions of DNA repair and enzyme photoactivation is a noncovalently bound FAD. In this paper, the FAD cofactor of Escherichia coli DNA photolyase was characterized as the neutral flavin semiquinone by EPR spectroscopy at 9.68 and 94.5 GHz. From the high-frequency/high-field EPR spectrum, the principal values of the axially symmetric g-matrix of FADH(*) were extracted. Both EPR spectra show an emerging hyperfine splitting of 0.85 mT that could be assigned to the isotropic hyperfine coupling constant (hfc) of the proton at N(5). To obtain more information about the electron spin density distribution ENDOR and TRIPLE resonance spectroscopies were applied. All major proton hfc's could be measured and unambiguously assigned to molecular positions at the isoalloxazin moiety of FAD. The isotropic hfc's of the protons at C(8alpha) and C(6) are among the smallest values reported for protein-bound neutral flavin semiquinones so far, suggesting a highly restricted delocalization of the unpaired electron spin on the isoalloxazin moiety. Two further hfc's have been detected and assigned to the inequivalent protons at C(1'). Some conclusions about the geometrical arrangement of the ribityl side chain with respect to the isoalloxazin ring could be drawn: Assuming tetrahedral angles at C(1') the dihedral angle between the C(1')-C(2') bond and the 2p(z)() orbital at N(10) has been estimated to be 170.4 degrees +/- 1 degrees.

Anisotropy↗
PubMed1999

Biosynthesis of a neo-epi-verrucosane diterpene in the liverwort Fossombronia alaskana. A retrobiosynthetic NMR study.

The biosynthesis of the diterpene 8alpha-acetoxy-13alpha-hydroxy-5-oxo-13-epi- neoverrucosane in the arctic liverwort Fossombronia alaskana was studied by incorporation experiments using [1-(13)C]- and [U-(13)C(6)]glucose as precursors. The (13)C-labeling patterns of acetyl-CoA, pyruvate, and phosphoenolpyruvate in intermediary metabolism were reconstructed from the (13)C NMR data of biosynthetic amino acids (leucine, alanine, phenylalanine) and were used to predict hypothetical labeling patterns for isopentenyl pyrophosphate formed via the mevalonate pathway and the deoxyxylulose pathway. The labeling patterns observed for the neoverrucosane diterpene were consistent with the intermediate formation of geranyllinaloyl pyrophosphate assembled from dimethylallyl pyrophosphate and three molecules of isopentenyl pyrophosphate generated predominantly or entirely via 1-deoxyxylulose 5-phosphate. The experimental data can be integrated into a detailed biosynthetic scheme involving a 1,5-hydride shift. The postulated involvement of the 1,5-hydride shift was confirmed by an incorporation experiment with [6,6-(2)H(2)]glucose.

Diterpenes↗
PubMed1999

Cytidine 5'-triphosphate-dependent biosynthesis of isoprenoids: YgbP protein of Escherichia coli catalyzes the formation of 4-diphosphocytidyl-2-C-methylerythritol.

2-C-methylerythritol 4-phosphate has been established recently as an intermediate of the deoxyxylulose phosphate pathway used for biosynthesis of terpenoids in plants and in many microorganisms. We show that an enzyme isolated from cell extract of Escherichia coli converts 2-C-methylerythritol 4-phosphate into 4-diphosphocytidyl-2-C-methylerythritol by reaction with CTP. The enzyme is specified by the hitherto unannotated ORF ygbP of E. coli. The cognate protein was obtained in pure form from a recombinant hyperexpression strain of E. coli harboring a plasmid with the ygbP gene under the control of a T5 promoter and lac operator. By using the recombinant enzyme, 4-diphosphocytidyl-[2-(14)C]2-C-methylerythritol was prepared from [2-(14)C]2-C-methylerythritol 4-phosphate. The radiolabeled 4-diphosphocytidyl-2-C-methylerythritol was shown to be efficiently incorporated into carotenoids by isolated chromoplasts of Capsicum annuum. The E. coli ygbP gene appears to be part of a small operon also comprising the unannotated ygbB gene. Genes with similarity to ygbP and ygbB are present in the genomes of many microorganisms, and their occurrence appears to be correlated with that of the deoxyxylulose pathway of terpenoid biosynthesis. Moreover, several microorganisms have genes specifying putative fusion proteins with ygbP and ygbB domains, suggesting that both the YgbP protein and the YgbB protein are involved in the deoxyxylulose pathway. A gene from Arabidopsis thaliana with similarity to ygbP carries a putative plastid import sequence, which is well in line with the assumed localization of the deoxyxylulose pathway in the plastid compartment of plants.

Amino Acid Sequence↗
PubMed1999

The intraflavin hydrogen bond in human electron transfer flavoprotein modulates redox potentials and may participate in electron transfer.

Electron-transfer flavoprotein (ETF) serves as an intermediate electron carrier between primary flavoprotein dehydrogenases and terminal respiratory chains in mitochondria and prokaryotic cells. The three-dimensional structures of human and Paracoccus denitrificans ETFs determined by X-ray crystallography indicate that the 4'-hydroxyl of the ribityl side chain of FAD is hydrogen bonded to N(1) of the flavin ring. We have substituted 4'-deoxy-FAD for the native FAD and investigated the analog-containing ETF to determine the role of this rare intra-cofactor hydrogen bond. The binding constants for 4'-deoxy-FAD and FAD with the apoprotein are very similar, and the energy of binding differs by only 2 kJ/mol. The overall two-electron oxidation-reduction potential of 4'-deoxy-FAD in solution is identical to that of FAD. However, the potential of the oxidized/semiquinone couple of the ETF containing 4'-deoxy-FAD is 0.116 V less than the oxidized/semiquinone couple of the native protein. These data suggest that the 4'-hydoxyl-N(1) hydrogen bond stabilizes the anionic semiquinone in which negative charge is delocalized over the N(1)-C(2)O region. Transfer of the second electron to 4'-deoxy-FAD reconstituted ETF is extremely slow, and it was very difficult to achieve complete reduction of the flavin semiquinone to the hydroquinone. The turnover of medium chain acyl-CoA dehydrogenase with native ETF and ETF containing the 4'-deoxy analogue was essentially identical when the reduced ETF was recycled by reduction of 2,6-dichlorophenolindophenol. However, the steady-state turnover of the dehydrogenase with 4'-deoxy-FAD was only 23% of the turnover with native ETF when ETF semiquinone formation was assayed directly under anaerobic conditions. This is consistent with the decreased potential of the oxidized semiquinone couple of the analog-containing ETF. ETF containing 4'-deoxy-FAD neither donates to nor accepts electrons from electron-transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) at significant rates (</=0.5% the wild-type rates). These results indicate that the 4'-hydroxyl-N(1) hydrogen bond plays a major role in the stabilization of the anionic semiquinone and anionic hydroquinone oxidation states of ETF and that this hydrogen bond may provide a pathway for electron transfer between the ETF flavin and the flavin of ETF-QO.

Animals↗
PubMed1999

NIH shift in flavin-dependent monooxygenation: mechanistic studies with 2-aminobenzoyl-CoA monooxygenase/reductase.

The flavoprotein 2-aminobenzoyl-CoA monooxygenase/reductase from the eubacterium Azoarcus evansii catalyzes the dearomatization of 2-aminobenzoyl-CoA. The reaction consists in an O2-dependent monooxygenation at the benzene position 5, which is followed immediately by an NADH-dependent hydrogenation of the intermediate at the same catalytic locus. The reaction was studied by 1H, 2H, and 13C NMR spectroscopy of the products. The main product was characterized as 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA by two-dimensional NMR spectroscopy. Thus, [5-2H]2-aminobenzoyl-CoA was converted into [6-2H]5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA, indicating a 5 --> 6 shift of the [5-2H] label. Label from NAD2H was transferred to the 3 position of the cyclic eneamine, whereas label from solvent D2O was incorporated into the 4 and the 6 positions of 5-oxo-2-aminocyclohex-1-ene-1-carboxyl-CoA. The labeling pattern is compatible with the monooxygenation proceeding via what is formally an NIH shift, yielding 5-oxo-2-aminocyclohex-1, 3-diene-1-carboxyl-CoA as a protein-bound intermediate. It is suggested that this shift in flavin-dependent monooxygenation may have general validity.

Acyl Coenzyme A↗
PubMed1999

Histidine 179 mutants of GTP cyclohydrolase I catalyze the formation of 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone triphosphate.

GTP cyclohydrolase I catalyzes the conversion of GTP to dihydroneopterin triphosphate. The replacement of histidine 179 by other amino acids affords mutant enzymes that do not catalyze the formation of dihydroneopterin triphosphate. However, some of these mutant proteins catalyze the conversion of GTP to 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone 5'-triphosphate as shown by multinuclear NMR analysis. The equilibrium constant for the reversible conversion of GTP to the ring-opened derivative is approximately 0.1. The wild-type enzyme converts the formylamino pyrimidine derivative to dihydroneopterin triphosphate; the rate is similar to that observed with GTP as substrate. The data support the conclusion that the formylamino pyrimidine derivative is an intermediate in the overall reaction catalyzed by GTP cyclohydrolase I.

Catalytic Domain↗
PubMed1999

Complementation of the fol2 deletion in Saccharomyces cerevisiae by human and Escherichia coli genes encoding GTP cyclohydrolase I.

Saccharomyces cerevisiae is so far the only organism where a knock-out mutant in the gene encoding GTP cyclohydrolase I (FOL2) has been obtained. GTP cyclohydrolase I controls the de novo biosynthetic pathway of tetrahydrobiopterin and folic acid. Since deletion of yeast FOL2 leads to a recessive auxotrophy for folinic acid, we used a yeast fol2Delta mutant for an in vivo functional assay of heterologous GTP cyclohydrolases I. We show that the GTP cyclohydrolase I, encoded either by the E. coli folE gene or by the human cDNA, complements the yeast fol2Delta mutation by restoring folate prototrophy. Furthermore the folE-3x allele of the E. coli gene, carrying three base substitutions, failed to complement the yeast fol2Delta defect. This allele behaved as a negative semidominant to the wild type folE and, when overexpressed, completely abolished complementation of fol2Delta by folE. Thus, the yeast fol2 null mutant is a suitable system to characterize mutations in genes encoding GTP cyclohydrolase I.

Alleles↗
PubMed1999

Dimethylallyl pyrophosphate is not the committed precursor of isopentenyl pyrophosphate during terpenoid biosynthesis from 1-deoxyxylulose in higher plants.

Cell cultures of Catharanthus roseus were supplied with [2-13C, 3-2H]-deoxyxylulose or [2-13C,4-2H]1-deoxyxylulose. Lutein and chlorophylls were isolated from the cell mass, and hydrolysis of the chlorophyll mixtures afforded phytol. Isotope labeling patterns of phytol and lutein were determined by 2H NMR and 1H,2H-decoupled 13C NMR. From the data it must be concluded that the deuterium atom in position 3 of deoxyxylulose was incorporated into both isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate with a rate of 75% (with respect to the internal 13C label). The detected stereochemical signature implies that the label is located preferentially in the (E)-hydrogen atom of IPP. This preferential labeling, in turn, rules out dimethylallyl pyrophosphate as the compulsory precursor of IPP. In the experiment with [2-13C, 4-2H]1-deoxyxylulose, the 13C label was efficiently transferred to the terpenoids whereas the 2H label was completely washed out, most probably after IPP formation as a consequence of the isomerization and elongation process. In addition, the data cast light on the stereochemical course of the dehydrogenation and cyclization steps involved in the biosynthesis of lutein.

Journal Article↗