Molecular recognition of aldehydes by aldehyde dehydrogenase and mechanism of nucleophile activation

Experimental structural data on the state of substrates bound to class 3 Aldehyde Dehydrogenases (ALDH3A1) is currently unknown. We have utilized molecular mechanics (MM) simulations, in conjunction with new force field parameters for aldehydes, to study the atomic details of benzaldehyde binding to ALDH3A1. Our results indicate that while the nucleophilic Cys243 must be in the neutral state to form what are commonly called near-attack conformers (NACs), these structures do not correlate with increased complexation energy calculated with the MM-Generalized Born Molecular Volume (GBMV) method. The negatively charged Cys243 (thiolate form) of ALDH3A1 also binds benzaldehyde in a stable conformation but in this complex the sulfur of Cys243 is oriented away from benzaldehyde yet yields the most favorable MM-GBMV complexation energy. The identity of the general base, Glu209 or Glu333, in ALDHs remains uncertain. The MM simulations reveal structural and possible functional roles for both Glu209 and Glu333. Structures from the MM simulations that would support either glutamate residue as the general base were further examined with Hybrid Quantum Mechanical (QM)/MM simulations. These simulations show that, with the PM3/OPLS potential, Glu209 must go through a step-wise mechanism to activate Cys243 through an intervening water molecule while Glu333 can go through a more favorable concerted mechanism for the same activation process.     

Sphingosine-1-phosphate plays a role in the suppression of lateral pseudopod formation during Dictyostelium discoideum cell migration and chemotaxis

Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that plays a role in diverse biological processes. It functions both as an extracellular ligand through a family of high-affinity G-protein-coupled receptors, and intracellularly as a second messenger. A growing body of evidence has implicated S-1-P in controlling cell movement and chemotaxis in cultured mammalian cells. Mutant D. discoideum cells, in which the gene encoding the S-1-P lyase had been specifically disrupted by homologous recombination, previously were shown to be defective in pseudopod formation, suggesting that a resulting defect might exist in motility and/or chemotaxis. To test this prediction, we analyzed the behavior of mutant cells in buffer, and in both spatial and temporal gradients of the chemoattractant cAMP, using computer-assisted 2-D and 3-D motion analysis systems. Under all conditions, S-1-P lyase null mutants were unable to suppress lateral pseudopod formation like wild-type control cells. This resulted in a reduction in velocity in buffer and spatial gradients of cAMP. Mutant cells exhibited positive chemotaxis in spatial gradients of cAMP, but did so with lowered efficiency, again because of their inability to suppress lateral pseudopod formation. Mutant cells responded normally to simulated temporal waves of cAMP but mimicked the temporal dynamics of natural chemotactic waves. The effect must be intracellular since no homologs of the S-1-P receptors have been identified in the Dictyostelium genome. The defects in the S-1-P lyase null mutants were similar to those seen in mutants lacking the genes for myosin IA, myosin IB, and clathrin, indicating that S-1-P signaling may play a role in modulating the activity or organization of these cytoskeletal elements in the regulation of lateral pseudopod formation.     

Hydrophobic complementarity in protein-protein docking

Formation of hydrophobic contacts across a newly formed interface is energetically favorable. Based on this observation we developed a geometric-hydrophobic docking algorithm that estimates quantitatively the hydrophobic complementarity at protein-protein interfaces. Each molecule to be docked is represented as a grid of complex numbers, storing information regarding the shape of the molecule in the real part and information regarding the hydropathy of the surface in the imaginary part. The grid representations are correlated using fast Fourier transformations. The algorithm is used to compare the extent of hydrophobic complementarity in oligomers (represented by D2 tetramers) and in hetero-dimers of soluble proteins (complexes). We also test the implication of hydrophobic complementarity in distinguishing correct from false docking solutions. We find that hydrophobic complementarity at the interface exists in oligomers and in complexes, and in both groups the extent of such complementarity depends on the size of the interface. Thus, the non-polar portions of large interfaces are more often juxtaposed than non-polar portions of small interfaces. Next we find that hydrophobic complementarity helps to point out correct docking solutions. In oligomers it significantly improves the ranks of nearly correct reassembled and modeled tetramers. Combining geometric, electrostatic and hydrophobic complementarity for complexes gives excellent results, ranking a nearly correct solution < 10 for 5 of 23 tested systems, < 100 for 8 systems and < 1000 for 19 systems.     

Dissecting physiological roles of estrogen receptor alpha and beta with potent selective ligands from structure-based design

The distinct roles of the two estrogen receptor (ER) isotypes, ERalpha and ERbeta, in mediating the physiological responses to estrogens are not completely understood. Although knockout animal experiments have been aiding to gain insight into estrogen signaling, additional information on the function of ERalpha and ERbeta will be provided by the application of isotype-selective ER agonists. Based on the crystal structure of the ERalpha ligand binding domain and a homology model of the ERbeta-ligand binding domain, we have designed steroidal ligands that exploit the differences in size and flexibility of the two ligand binding cavities. Compounds predicted to bind preferentially to either ERalpha or ERbeta were synthesized and tested in vitro using radio-ligand competition and transactivation assays. This approach directly led to highly ER isotype-selective (approximately 200-fold) and potent ligands. To unravel physiological roles of the two receptors, in vivo experiments with rats were conducted using the ERalpha- and ERbeta-selective agonists in comparison to 17beta-estradiol. The ERalpha agonist induced uterine growth, caused bone-protective effects, reduced LH and FSH plasma levels, and increased angiotensin I, whereas the ERbeta agonist did not at all or only at high doses lead to such effects, despite high plasma levels. It can thus be concluded that estrogen effects on the uterus, pituitary, bone, and liver are primarily mediated via ERalpha. Simultaneous administration of the ERalpha and ERbeta ligand did not lead to an attenuation of ERalpha-mediated effects on the uterus, pituitary, and liver parameters.     

Htd2p/Yhr067p is a yeast 3-hydroxyacyl-ACP dehydratase essential for mitochondrial function and morphology

Among the recently recognized aspects of mitochondrial functions, in yeast as well as humans, is their ability to synthesize fatty acids in a malonyl-CoA dependent manner. We describe here the identification of the 3-hydroxyacyl-ACP dehydratase involved in mitochondrial fatty acid synthesis. A colony-colour-sectoring screen was applied in Saccharomyces cerevisiae in a search for mutants that, when grown on a non-fermentable carbon source, were unable to lose a plasmid that carried a chimeric construct coding for mitochondrially localized bacterial analogue. Our mutants, which are respiratory deficient, lack cytochromes and display abnormal mitochondrial morphology, were found to have a lesion in the yeast YHR067w/RMD12 gene. The Yhr067p is predicted to be a member of the thioesterase/thioester dehydratase-isomerase superfamily enzymes. Hydratase 2 activity in mitochondrial extracts from cells overexpressing YHR067w was increased. These overexpressing cells also display a striking mitochondrial enlargement phenotype. We conclude that YHR067w encodes a novel mitochondrial 3-hydroxyacyl-thioester dehydratase 2 and suggest renaming it HTD2. The mitochondrial phenotypes of the null and overexpression mutants suggest a crucial role of YHR067w in maintenance of mitochondrial respiratory competence and morphology in yeast.     

The folate pathway is a target for resistance to the drug para-aminosalicylic acid (PAS) in mycobacteria

The increasing rate of multidrug-resistant tuberculosis has led to more use of second-line antibiotics such as para-aminosalicylic acid (PAS). The mode of action of PAS remains unclear, and mechanisms of resistance to this drug are undefined. We have isolated PAS-resistant transposon mutants of Mycobacterium bovis BCG with insertions in the thymidylate synthase (thyA) gene, a critical determinant of intracellular folate levels. BCG thyA mutants have reduced thymidylate synthase activity and are resistant to known inhibitors of the folate pathway. We also find that mutations in thyA are associated with clinical PAS resistance. We have identified PAS-resistant Mycobacterium tuberculosis isolates from infected patients, which harbour mutations in thyA and show reduced activity of the encoded enzyme. Thus, PAS acts in the folate pathway, and thyA mutations probably represent a mechanism of developing resistance not only to PAS but also to other drugs that target folate metabolism.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

Phosphoinositide-specific phospholipase C is involved in cytokinin and gravity responses in the moss Physcomitrella patens

The phosphoinositide signalling pathway is important in plant responses to extracellular and intracellular signals. To elucidate the physiological functions of phosphoinositide-specific phopspholipase C, PI-PLC, targeted knockout mutants of PpPLC1, a gene encoding a PI-PLC from the moss Physcomitrella patens, were generated via homologous recombination. Protonemal filaments of the plc1 lines show a dramatic reduction in gametophore formation relative to wild type: this was accompanied by a loss of sensitivity to cytokinin. Moreover, plc1 appeared paler than the wild type, the result of an altered differentiation of chloroplasts and reduced chlorophyll levels compared with wild type filaments. In addition, the protonemal filaments of plc1 have a strongly reduced ability to grow negatively gravitropically in the dark. These effects imply a significant role for PpPLC1 in cytokinin signalling and gravitropism.     

Increased heat loss affects hibernation in golden-mantled ground squirrels

During hibernation at ambient temperatures (T(a)) above 0 degrees C, rodents typically maintain body temperature (T(b)) approximately 1 degrees C above T(a), reduce metabolic rate, and suspend or substantially reduce many physiological functions. We tested the extent to which the presence of an insulative pelage affects hibernation. T(b) was recorded telemetrically in golden-mantled ground squirrels (Spermophilus lateralis) housed at a T(a) of 5 degrees C; food intake and body mass were measured at regular intervals throughout the hibernation season and after the terminal arousal. Animals were subjected to complete removal of the dorsal fur or a control procedure after they had been in hibernation for 3-4 wk. Shaved squirrels continued to hibernate with little or no change in minimum T(b), bout duration, duration of periodic normothermic bouts, and food intake during normothermia. Rates of rewarming from torpor were, however, significantly slower in shaved squirrels, and rates of body mass loss were significantly higher, indicating increased depletion of white adipose energy stores. An insulative pelage evidently conserves energy over the course of the hibernation season by decreasing body heat loss and reducing energy expenditure during periodic arousals from torpor and subsequent intervals of normothermia. This prolongs the hibernation season by several weeks, thereby eliminating the debilitating consequences associated with premature emergence from hibernation.