Graham E. Ball

Regulation of the balance of one-carbon metabolism in Saccharomyces cerevisiae.

One-carbon metabolism in yeast is an essential process that relies on at least one of three one-carbon donor molecules: serine, glycine, or formate. By a combination of genetics and biochemistry we have shown how cells regulate the balance of one-carbon flow between the donors by regulating cytoplasmic serine hydroxymethyltransferase activity in a side reaction occurring in the presence of excess glycine. This control governs the level of 5,10-methylene tetrahydrofolate (5,10-CH2-H4folate) in the cytoplasm, which has a direct role in signaling transcriptional control of the expression of key genes, particularly those encoding the unique components of the glycine decarboxylase complex (GCV1,GCV2, and GCV3). Based on these and other observations, we propose a model for how cells balance the need to supplement their one-carbon pools when charged folates are limiting or when glycine is in excess. We also propose that under normal conditions, cytoplasmic 5,10-CH2-H4folate is mainly directed to generating methyl groups via methionine, whereas one-carbon units generated from glycine in mitochondria are more directed to purine biosynthesis. When glycine is in excess, 5,10-CH2-H4folate is decreased, and the regulation loop shifts the balance of generation of one-carbon units into the mitochondrion.

NMR and modelling studies of disaccharide conformation

Long-range heteronuclear coupling constants were measured across the glycosidic linkages for a series of eight alpha- or beta-linked disaccharides in aqueous solution. Multiple 13C site-selective excitation experiments using 1H decoupling in conjunction with pulsed field gradient-enhanced spectroscopy were used to determine 3J(C,H) values. These were subsequently compared with the respective couplings calculated, using a Karplus relationship, from molecular dynamics simulations with the explicit inclusion of water.

Roles of α and β Carbonic Anhydrases of Helicobacter pylori in the Urease-Dependent Response to Acidity and in Colonization of the Murine Gastric Mucosa

ABSTRACT Carbon dioxide occupies a central position in the physiology of Helicobacter pylori owing to its capnophilic nature, the large amounts of carbon dioxide produced by urease-mediated urea hydrolysis, and the constant bicarbonate supply in the stomach. Carbonic anhydrases (CA) catalyze the interconversion of carbon dioxide and bicarbonate and are involved in functions such as CO2 transport or trapping and pH homeostasis. H. pylori encodes a periplasmic α-CA (α-CA-HP) and a cytoplasmic β-CA (β-CA-HP). Single CA inactivation and double CA inactivation were obtained for five genetic backgrounds, indicating that H. pylori CA are not essential for growth in vitro. Bicarbonate-carbon dioxide exchange rates were measured by nuclear magnetic resonance spectroscopy using lysates of parental strains and CA mutants. Only the mutants defective in the α-CA-HP enzyme showed strongly reduced exchange rates. In H. pylori, urease activity is essential for acid resistance in the gastric environment. Urease activity measured using crude cell extracts was not modified by the absence of CA. With intact CA mutant cells incubated in acidic conditions (pH 2.2) in the presence of urea there was a delay in the increase in the pH of the incubation medium, a phenotype most pronounced in the absence of H. pylori α-CA. This correlated with a delay in acid activation of the urease as measured by slower ammonia production in whole cells. The role of CA in vivo was examined using the mouse model of infection with two mouse-adapted H. pylori strains, SS1 and X47-2AL. Compared to colonization by the wild-type strain, colonization by X47-2AL single and double CA mutants was strongly reduced. Colonization by SS1 CA mutants was not significantly different from colonization by wild-type strain SS1. However, when mice were infected by SS1 Δ(β-CA-HP) or by a SS1 double CA mutant, the inflammation scores of the mouse gastric mucosa were strongly reduced. In conclusion, CA contribute to the urease-dependent response to acidity of H. pylori and are required for high-grade inflammation and efficient colonization by some strains.

A delicate balance of complexation vs. activation of alkanes interacting with [Re(Cp)(CO)(PF3)] studied with NMR and time-resolved IR spectroscopy

The organometallic alkane complexes Re(Cp)(CO)(PF3)(alkane) and Re(Cp)(CO)2(alkane) have been detected after the photolysis of Re(Cp)(CO)2(PF3) in alkane solvent. NMR and time-resolved IR experiments reveal that the species produced by the interaction of n-pentane with [Re(Cp)(CO)(PF3)] are an equilibrium mixture of Re(Cp)(CO)(PF3)(pentane) and Re(Cp)(CO)(PF3)(pentyl)H. The interaction of cyclopentane with [Re(Cp)(CO)(PF3)] most likely results in a similar equilibrium between cyclopentyl hydride and cyclopentane complexes. An increasing proportion of alkane complex is observed on going from n-pentane to cyclopentane to cyclohexane, where only a small amount, if any, of the cyclohexyl hydride form is present. In general, when [Re(Cp)(CO)(PF3)] reacts with alkanes, the products display a higher degree of oxidative cleavage in comparison with [Re(Cp)(CO)2], which favors alkane complexation without activation. Species with the formula Re(Cp)(CO)(PF3)(alkane) have higher thermal stability and lower reactivity toward CO than the analogous Re(Cp)(CO)2(alkane) complexes.

Pyruvate metabolism in Campylobacter spp.

The metabolism of pyruvate by Campylobacter spp. was investigated employing one- and two-dimensional 1H, 13C and 31P nuclear magnetic resonance spectroscopy. Metabolically competent cells incubated aerobically with pyruvate yielded acetate, acetolactate, alanine, formate, lactate, and succinate. The production of acetolactate, alanine and lactate indicated the presence of acetohydroxy acid synthase, alanine transaminase and lactate dehydrogenase activities, respectively. Accumulation of acetate and formate as metabolic products provided evidence for the existence of a mixed acid fermentation pathway in the microorganism. Formation of succinate suggested the incorporation of the pyruvate carbon skeleton to the Krebs cycle, and the observation of pyruvate dehydrogenase activities in bacterial lysates supported this interpretation. Generation of pyruvate from L-serine in incubations with intact cells and lysates indicated the presence of serine dehydratase activity in the bacterium. Pyruvate was also formed in cell suspensions and lysates from phosphoenol pyruvate. The existence of anaplerotic sequences involving phosphoenol pyruvate carboxykinase and a malic enzyme were established in bacterial lysates. The activities of enzymes involved in the biosynthesis of isoleucine and valine were measured. Addition of pyruvate to different solid culture media inhibited bacterial growth, and the inhibition was attributed to the accumulation of acetate and formate. The variety of products formed using pyruvate as the sole substrate and the existence of anaplerotic sequences and anabolic pathways which employ pyruvate, showed the important role of this metabolite in the energy and biosynthesis metabolism of Campylobacter spp.

Modulation of Brain Metabolism by Very Low Concentrations of the Commonly Used Drug Delivery Vehicle Dimethyl Sulfoxide (DMSO)

Dimethyl sulfoxide (DMSO) has long been used in studies as a vehicle to enhance the solubility and transport of ligands in biological systems. The effects of this drug on the outcomes of such studies are still unclear, with concentrations of DMSO reported as “safe” varying considerably. In the present work, we investigated the effects of very low concentrations of DMSO on the brain metabolism of [3‐13C]pyruvate and D‐[1‐13C]glucose using 1H/13C NMR spectroscopy and a guinea pig cortical brain slice model. Our results show that DMSO is accumulated by brain slices. DMSO at all concentrations [0.000025%–0.25% (v/v)] increased the metabolic rate when [3‐13C]pyruvate was used as a substrate and also in the presence of D‐[1‐13C]glucose (0.00025%–0.1% DMSO). These results are consistent with DMSO stimulating respiration, which it may do through altering the kinetics of ATP‐requiring reactions. Our results also emphasize that there is no practical concentration of DMSO that can be used in metabolic experiments without effect. Therefore, care should be taken when evaluating the actions of drugs administered in combination with DMSO.

Photoinduced N2 loss as a route to long-lived organometallic alkane complexes: A time-resolved IR and NMR study

Photolysis of CpRe(CO)2(N2) in cyclopentane or 2,2-dimethylbutane with a UV lamp via a quartz fibre inserted into the NMR probe allows generation of CpRe(CO)2(cyclopentane) and CpRe(CO)2(2,2-dimethylbutane). The latter is observed in three isomeric forms according to the site of co-ordination to the rhenium. The major isomer, CpRe(CO)2(2,2-dimethylbutane-η2-C1,H1), exhibits a 1H NMR resonance for the co-ordinated hydrogen at δ = −2.19 with 1JC–H = 118 Hz. The photochemistry of Cp‡Re(CO)2(N2) (Cp‡ = η5-1,2-C5H3(tBu)2) in alkane solution is also reported. Two new organometallic alkane complexes, Cp‡Re(CO)2(alkane) (alkane = cyclopentane, n-heptane) have been characterized by IR spectroscopy following irradiation of Cp‡Re(CO)2(N2) and their rate constants for reaction with CO have been determined. The reaction with cyclopentane has also been studied by NMR spectroscopy at 190 K with in situ laser irradiation at 355 nm. Cp‡Re(CO)2(c-C5H10) is shown to exhibit the characteristic features of an alkane complex in the NMR spectrum, viz. a large isotopic shift of the 1H resonance at δ = −2.44 upon partial deuteration of the alkane (Δδ = 1.77 ppm), a large 1JC–H (114 Hz) and a large negative 13C chemical shift (δ = −33.8). We find no evidence for CO loss or agostic interactions of the t-butyl groups under these conditions. Cp‡Re(CO)2(alkane) has a slightly shorter lifetime (ca. 5x) than CpRe(CO)2(alkane) for a given alkane. Photolysis of CpRe(CO)2(N2) to form the organometallic alkane complex occurs with a much higher yield than for CpRe(CO)3. Efficient photo-ejection of N2 from Cp‡Re(CO)2(N2) is observed upon either 266 or 355 nm laser irradiation. A dinitrogen precursor allows for the use of longer wavelength irradiation and the generation of a higher concentration of the alkane complex following each laser pulse.

Observation of a Tungsten Alkane σ-Complex Showing Selective Binding of Methyl Groups Using FTIR and NMR Spectroscopies

The alkane σ-complex (HEB)W(CO)(2)(pentane) (HEB = η(6)-hexaethylbenzene) is produced from the UV photolysis of (HEB)W(CO)(3) in alkane solvents at low temperature. IR and (1)H and (13)C NMR spectroscopic data are reported, representing the first NMR data for a group 6 alkane complex. Only binding of the methyl functionality of the pentane ligand was observed in (HEB)W(CO)(2)(pentane). This contrasts with the previously reported binding of pentane to rhenium fragments, wherein both methylene and methyl groups were observed to bind, with a slight preference for binding of the former. The reason for the preference for binding through the methyl group is investigated, and the steric requirement for the pentane to adopt an unfavorable gauche conformation when bound via a methylene is identified as a contributing factor.

Transition metal-alkane σ-complexes with oxygen donor co-ligands

A new family of long-lived alkane σ-complexes of the type (L(OEt))Re(CO)(2)(alkane) [alkane = cyclopentane, cyclohexane, pentane; L(OEt) = cyclopentadienyltris(diethylphosphito)cobaltate(III)] has been observed using both IR and NMR spectroscopies and computationally interrogated with DFT methods. The oxygen-rich coordination spheres makes these complexes perhaps more relevant as models for intermediates in metal oxide mediated hydrocarbon transformations than other known alkane σ-complexes.

A Benchmark Ab Initio and DFT Study of the Structure and Binding of Methane in the σ-Alkane Complex CpRe(CO)2(CH4).

Ab initio molecular orbital theory and density functional theory (DFT) procedures have been used to study the binding of methane in CpRe(CO)2(CH4), the simplest σ-alkane complex in the experimentally widely studied CpRe(CO)2(alkane) family. We find the optimal Re···C, Re···H and C···H distances to be 2.60, 1.92, and 1.15 Å, respectively, on the composite-CCSD(T)/def2-QZVPP (CCSD(T)/def2-TZVP with supplement for the larger def2-QZVPP basis set at the second-order Møller-Plesset perturbation theory level) potential energy surface which has been mapped out at this level of theory. The enthalpy of binding at 298 K was determined to be 62.0 kJ mol(-1) at the composite-CCSD(T)/CBS//B3-PW91/aug-cc-pVTZ-PP level. Benchmarks on the various DFT procedures show that some functionals give good geometries but underestimate binding energies, while others yield poor geometries but give closer agreements with the composite-CCSD(T) binding energy. On the other hand, the ωB97X-D functional gives fair agreements with composite-CCSD(T) for both geometry optimization as well as binding energy. Thus, it appears to be a reliable, easily implemented, and cost-effective means for studying Re-alkane complexes. Good binding energies are also obtained with several common functionals when D3 dispersion corrections are applied. Selected dispersion-corrected DFT methods (B3PW91-D3, TPSSh-D3, and B98-D3) were found to be quite accurate for the calculation of binding energies of several other model metal-CH4 complexes containing a range of metal centers (Rh, Pd, W, Ir, Pt). We also note that, for single-point energy calculation of the Re-CH4 binding, the PWP-B95-D3 double-hybrid DFT procedure provides an excellent agreement with the benchmark energy at only a slightly higher computational requirement.