13C-, 15N- and 31P-NMR studies of oxidized and reduced low molecular mass thioredoxin reductase and some mutant proteins.

Thioredoxin reductase (TrxR) from Escherichia coli, the mutant proteins E159Y and C138S, and the mutant protein C138S treated with phenylmercuric acetate were reconstituted with [U-(13)C(17),U-(15)N(4)]FAD and analysed, in their oxidized and reduced states, by (13)C-, (15)N- and (31)P-NMR spectroscopy. The enzymes studied showed very similar (31)P-NMR spectra in the oxidized state, consisting of two peaks at -9.8 and -11.

Exaggerated exercise blood pressure is related to impaired endothelial vasodilator function.

Background: Persons with high normal blood pressure (BP) or mild hypertension who also have an exaggerated BP response to exercise are at risk for worsening hypertension. The mechanisms that explain this relationship are unknown. We examined the relationships of endothelial vasodilator function and of aortic stiffness with exercise BP.

Design, synthesis, and evaluation of 9-D-ribitylamino-1,3,7,9-tetrahydro-2,6,8-purinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents as inhibitors of tiboflavin synthase and lumazine synthase.

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin, an essential metabolite that is involved in electron transport processes. To obtain structural probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a series of ribitylpurinetriones bearing alkyl phosphate and alpha,alpha-difluorophosphonate substituents were synthesized. Since the purinetrione ring system and the ribityl hydroxyl groups can be alkylated, the synthesis required the generation of these two moieties in protected form before the desired alkylation reaction could be carried out.

Riboflavin synthase of Schizosaccharomyces pombe. Protein dynamics revealed by 19F NMR protein perturbation experiments.

Background: Riboflavin synthase catalyzes the transformation of 6,7-dimethyl-8-ribityllumazine into riboflavin in the last step of the riboflavin biosynthetic pathway. Gram-negative bacteria and certain yeasts are unable to incorporate riboflavin from the environment and are therefore absolutely dependent on endogenous synthesis of the vitamin. Riboflavin synthase is therefore a potential target for the development of antiinfective drugs.

Therapeutic hypothermia.

Hypothermia is common during anaesthesia and surgery owing to anaesthetic-induced inhibition of thermoregulatory control. Perioperative hypothermia is associated with numerous complications. However, for certain patient populations, and under specific clinical conditions, hypothermia can provide substantial benefits.

Observation of the charge symmetry breaking d+d-->4He+pi(0) reaction near threshold.

We report the first observation of the charge symmetry breaking d+d-->4He+pi(0) reaction near threshold. Measurements using a magnetic channel (gated by two photons) of the 4He scattering angle and momentum (from time of flight) permitted reconstruction of the pi(0) "missing mass," the quantity used to separate 4He+pi(0) events from the continuum of double radiative capture 4He+gamma+gamma events. We measured total cross sections for neutral pion production of 12.

Presteady state kinetic analysis of riboflavin synthase.

Riboflavin synthase catalyzes a mechanistically complex dismutation affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione from 6,7-dimethyl-8-ribityllumazine. The kinetics of the enzyme from Escherichia coli were studied under single turnover conditions. Stopped flow as well as quenched flow experiments documented the transient formation of a pentacyclic reaction intermediate.

Biosynthetic origin of a branched chain analogue of the lipase inhibitor, lipstatin.

The lipophilic beta-lactone, lipstatin, inhibits pancreatic lipase and has been shown earlier to be biosynthesized by Claisen condensation of two fatty acid moieties. We present data from incorporation experiments with [U-13C6]leucine showing that a branched chain analogue of lipstatin is biosynthesized from leucine.

The structure of the N-terminal domain of riboflavin synthase in complex with riboflavin at 2.6A resolution.

Riboflavin synthase of Escherichia coli is a homotrimer with a molecular mass of 70 kDa. The enzyme catalyzes the dismutation of 6,7-dimethyl-8-(1'-D-ribityl)-lumazine, affording riboflavin and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The N-terminal segment (residues 1-87) and the C-terminal segment (residues 98-187) form beta-barrels with similar fold and a high degree of sequence similarity.

Structure and properties of an engineered transketolase from maize.

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK.

Structure of 3,4-dihydroxy-2-butanone 4-phosphate synthase from Methanococcus jannaschii in complex with divalent metal ions and the substrate ribulose 5-phosphate: implications for the catalytic mechanism.

Skeletal rearrangements of carbohydrates are crucial for many biosynthetic pathways. In riboflavin biosynthesis ribulose 5-phosphate is converted into 3,4-dihydroxy-2-butanone 4-phosphate while its C4 atom is released as formate in a sequence of metal-dependent reactions. Here, we present the crystal structure of Methanococcus jannaschii 3,4-dihydroxy-2-butanone 4-phosphate synthase in complex with the substrate ribulose 5-phosphate at a dimetal center presumably consisting of non-catalytic zinc and calcium ions at 1.

Biosynthesis of isoprenoids: crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase.

4-Diphosphocytidyl-2C-methyl-d-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate. The 2-A resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the alpha/beta fold characteristic of the galactose kinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains.

Temperature-dependent presteady state kinetics of lumazine synthase from the hyperthermophilic eubacterium Aquifex aeolicus.

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Presteady state kinetic experiments using the enzyme from the hyperthermophilic bacterium Aquifex aeolicus were monitored by multiwavelength photometry. An early optical transient absorbing around 330 nm is interpreted as a Schiff base intermediate obtained by reaction of the position 5 amino group of the heterocyclic substrate with the carbonyl group of 3,4-dihydroxy-2-butanone 4-phosphate.

Functional genomics by NMR spectroscopy. Phenylacetate catabolism in Escherichia coli.

Aerobic metabolism of phenylalanine in most bacteria proceeds via oxidation to phenylacetate. Surprisingly, the further metabolism of phenylacetate has not been elucidated, even in well studied bacteria such as Escherichia coli. The only committed step is the conversion of phenylacetate into phenylacetyl-CoA.

Phenylalanine-independent biosynthesis of 1,3,5,8-tetrahydroxyxanthone. A retrobiosynthetic NMR study with root cultures of Swertia chirata.

Root cultures of Swertia chirata (Gentianaceae) were grown with supplements of [1-13C]glucose, [U-13C6]glucose or [carboxy-13C]shikimic acid. 1,3,5,8-Tetrahydroxyxanthone was isolated and analysed by quantitative NMR analysis. The observed isotopomer distribution shows that 1,3,5,8-tetrahydroxyxanthone is biosynthesized via a polyketide-type pathway.

Crystal structure of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Bacillus subtilis.

Two types of isopentenyl diphosphate:dimethylallyl diphosphate isomerases (IDI) have been characterized at present. The long known IDI-1 is only dependent on divalent metals for activity, whereas IDI-2 requires a metal, FMN and NADPH. Here, we report the first structure of an IDI-2 from Bacillus subtilis at 1.

Exercise blood pressure response is related to left ventricular mass.

An exaggerated SBP response to exercise has been associated with increased left ventricular (LV) mass in some but not all studies. A total of 43 women and 34 men, aged 55-75 years, without evidence of cardiovascular disease, with a mean resting BP of 142+/-9/77+/-8 mmHg had their BP measured at rest and during maximal treadmill exercise. LV mass was measured using magnetic resonance imaging.

A structure-based model of the reaction catalyzed by lumazine synthase from Aquifex aeolicus.

6,7-Dimethyl-8-ribityllumazine is the biosynthetic precursor of riboflavin, which, as a coenzyme, plays a vital role in the electron transfer process for energy production in all cellular organisms. The enzymes involved in lumazine biosynthesis have been studied in considerable detail. However, the conclusive mechanism of the reaction catalyzed by lumazine synthase has remained unclear.

Biosynthesis of riboflavin. Single turnover kinetic analysis of 6,7-dimethyl-8-ribityllumazine synthase.

6,7-dimethyl-8-ribityllumazine synthase (lumazine synthase) catalyzes the condensation of 5-amino-6-ribitylamino-2,4-(1H,3H)-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate, affording the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine. Single turnover experiments monitored by multiwavelength photometry were performed with the recombinant lumazine synthase of Bacillus subtilis. Mixing of the enzyme with the pyrimidine type substrate is conducive to a hypsochromic shift as well as a decrease in absorbance of the heterocyclic substrate; the rate constant for that reaction is 0.

Are fitness, activity, and fatness associated with health-related quality of life and mood in older persons?

Purpose: This study sought to determine whether levels of fitness, habitual physical activity, and fatness are associated health-related quality of life and mood in older persons.

Methods: The subjects were men (n = 38) and women (n = 44), ages 55 to 75 years, who had milder forms of hypertension, but who were otherwise healthy and not engaged in a regular exercise or diet program. Aerobic fitness was assessed by maximal oxygen uptake during treadmill testing, muscle strength by a one-repetition maximum, habitual activity by questionnaire, fatness by dual-energy x-ray absorptiometry, and body mass index.

Usefulness of anthropometrics and dual-energy x-ray absorptiometry for estimating abdominal obesity measured by magnetic resonance imaging in older men and women.

Purpose: Increasing evidence suggests that abdominal obesity may be a better predictor of disease risk than total fatness. This study sought to determine how obesity and fat distribution measured by readily available anthropometric and dual-energy x-ray absorptiometry (DXA) methods is related to abdominal obesity assessed by magnetic resonance imaging (MRI).

Methods: Men (n = 43) and women (n = 47), ages 55 to 75 years, were assessed for body mass index, waist-to-hip ratio, waist circumference, and skin folds by anthropometric methods; for percentage of body fat by DXA; and for abdominal total, subcutaneous, and visceral fat by MRI.

RNA DNA discrimination by the antitermination protein NusB.

The regulation of ribosomal RNA biosynthesis in Escherichia coli by antitermination requires binding of NusB protein to a dodecamer sequence designated boxA on the nascent RNA. The affinity of NusB protein for boxA RNA exceeds that for the homologous DNA segment by more than three orders of magnitude as shown by surface plasmon resonance measurements. DNA RNA discrimination by NusB protein was shown to involve methyl groups (i.

Evidence for a strong surface-plasmon resonance on ErAs nanoparticles in GaAs.

Room-temperature attenuation measurements are made between lambda=0.8 and 10.0 microm on three GaAs epitaxial samples containing layers of ErAs nanoparticles.

Structural basis of fosmidomycin action revealed by the complex with 2-C-methyl-D-erythritol 4-phosphate synthase (IspC). Implications for the catalytic mechanism and anti-malaria drug development.

2-C-Methyl-d-erythritol 4-phosphate synthase (IspC) is the first enzyme committed to isoprenoid biosynthesis in the methylerythritol phosphate pathway, which represents an alternative route to the classical mevalonate pathway. As it is present in many pathogens and plants, but not in man, this pathway has attracted considerable interest as a target for novel antibiotics and herbicides. Fosmidomycin represents a specific high-affinity inhibitor of IspC.

Biosynthesis of riboflavin in archaea. 6,7-dimethyl-8-ribityllumazine synthase of Methanococcus jannaschii.

Heterologous expression of the putative open reading frame MJ0303 of Methanococcus jannaschii provided a recombinant protein catalysing the formation of the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, by condensation of 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and 3,4-dihydroxy-2-butanone 4-phosphate. Steady state kinetic analysis at 37 degrees C and pH 7.0 indicated a catalytic rate of 11 nmol.