Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase

A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.     

Explicit water near the catalytic I helix Thr in the predicted solution structure of CYP2A4

The solution structure of mouse cytochrome P450 2A4 (CYP2A4), a monooxygenase of deoxysteroids, was obtained using homology modeling and molecular dynamics. The solvent-equilibrated CYP2A4 preserves the essential features of CYP450s. A comparison of the models CYP2A4 and CYP2A4 with testosterone bound CYP2A4/T illustrates the changes induced by the binding of the substrate. Experimental evidence links four amino acid residues to the catalytic activity, substrate specificity, and regioselectivity of this enzyme. Three of the four amino acids are found within contact distance of the testosterone substrate, and therefore may control the binding of the substrate through direct interaction. Remarkably, a water complex previously observed in x-ray crystal structure forms near the bulge in the central I helix that contains a conserved Thr. The properties of the I helix are computed in the context of the presence or absence of ligand.     

Glucose metabolism in cancer. Evidence that demethylation events play a role in activating type II hexokinase gene expression

One of the "signature" phenotypes of highly malignant, poorly differentiated tumors, including hepatomas, is their remarkable propensity to utilize glucose at a much higher rate than normal cells, a property frequently dependent on the marked overexpression of type II hexokinase (HKII). As the expression of the gene for this enzyme is nearly silent in liver tissue, we tested the possibility that DNA methylation/demethylation events may be involved in its regulation. Initial studies employing methylation restriction endonuclease analysis provided evidence for differential methylation patterns for the HKII gene in normal hepatocytes and hepatoma cells, the latter represented by a highly glycolytic model cell line (AS-30D). Subsequently, sequencing following sodium bisulfite treatment revealed 18 methylated CpG sites within a CpG island (-350 to +781 bp) in the hepatocyte gene but none in that of the hepatoma. In addition, treatment of a hepatocyte cell line with the DNA methyltransferase inhibitors, 5'-azacytidine and 5'-aza-2'-deoxycytidine, activated basal expression levels of HKII mRNA and protein. Finally, stably transfecting the hepatocyte cell line with DNA demethylase also resulted in activating the basal expression levels of HKII mRNA and protein. These novel observations indicate that one of the initial events in activating the HKII gene during either transformation or tumor progression may reside at the epigenetic level.     

Two mutants of human heparin binding protein (CAP37): toward the understanding of the nature of lipid A/LPS and BPTI binding

Heparin binding protein (HBP) is an inactive serine protease homologue with important implications in host defense during infections and inflammations. Two mutants of human HBP, [R23S,F25E]HBP and [G175Q]HBP, have been produced to investigate structure-function relationships of residues in the putative lipid A/lipopolysaccharide (LPS) binding site and BPTI (bovine pancreatic trypsin inhibitor) binding site. The X-ray structures have been determined at 1.9 A resolution for [G175Q]HBP and at 2.5 A resolution for the [R23S,F25E]HBP mutant, and the structures have been fully refined to R-factors of 18.2 % and 20.7 %, respectively. The G175Q mutation does not alter the overall structure of the protein, but the ability to bind BPTI has been eliminated, and the mutant mediates only a limited stimulation of the LPS-induced cytokine release from human monocytes. The lipid A/LPS binding property of [G175Q]HBP is comparable with that of native HBP. The R23S,F25E mutations do not affect the binding of lipid A/LPS and BPTI or the LPS-induced cytokine release from human monocytes. This shows that two diverse ligands, lipid A/LPS and BPTI, do not share binding sites. Previously, there was convincing evidence for the proposed lipid A/LPS binding site of HBP. Unexpectedly, the extensive structural changes introduced by mutation of Arg23 and Phe25 do not affect the binding of lipid A/LPS, indicating that another not yet identified site on HBP is involved in the binding of lipid A/LPS.     

The origin of the ankyrin repeat region in Notch intracellular domains is critical for regulation of HES promoter activity

Notch signal transduction is mediated by proteolysis of the receptor and translocation of the intracellular domain (IC) into the nucleus, where it functions as a regulator of HES gene expression after binding to the DNA-binding protein RBP-J kappa. The mammalian Notch receptors are structurally very similar, but have distinct functions. Most notably, Notch 1 IC is a potent activator of the HES promoter, while Notch 3 IC is a much weaker activator and can repress Notch 1 IC-mediated HES activation in certain contexts. In this report we explore the molecular basis for this functional difference between Notch 1 and Notch 3 IC. We find that Notch 3 IC, like Notch 1 IC, can bind the SKIP and PCAF proteins. Furthermore, both Notch 1 and Notch 3 ICs displace the co-repressor SMRT from the DNA-binding protein RBP-J kappa on the HES promoter. The latter observation suggests that both Notch 3 IC and Notch 1 IC can access RBP-J kappa in vivo, and that the difference in activation capacity instead stems from structural differences in the two ICs when positioned on RBP-J kappa. We show that two distinct regions in the Notch IC are critical for the difference between the Notch 1 and Notch 3 IC. First, the origin of the ankyrin repeat region is important, i.e. only chimeric ICs containing a Notch 1-derived ankyrin repeat region are potent activators. Second, we identify a novel important region in the Notch IC. This region, named the RE/AC region (for repression/activation), is located immediately C-terminal to the ankyrin repeat region, and is required for Notch 1 IC's ability to activate and for Notch 3 IC's ability to repress a HES promoter. The interplay between the RE/AC region and the ankyrin repeat region provides a basis to understand the difference in HES activation between structurally similar Notch receptors.     

Expression of genes encoding F(1)-ATPase results in uncoupling of glycolysis from biomass production in Lactococcus lactis

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F(1) domain of the membrane-bound (F(1)F(0)) H(+)-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F(1)-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F(1)-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.     

Directed evolution of barnase stability using proteolytic selection

We report the construction of a phage-displayed repertoire of mutants of the ribonuclease barnase from Bacillus amyloliquefaciens. The construction was guided by the natural variability between two closely related ribonucleases, barnase and binase from Bacillus intermedius. This repertoire was selected using a proteolytic selection method, allowing sorting of the library according to the resistance of the mutants toward proteolysis. Susceptibility toward proteolysis has been correlated with flexibility and unfolding, and is thus expected to yield mutants with increased thermal stability.Enrichment of barnase mutants with specific combinations of amino acid residues at four of the randomised positions was observed. Three of these enriched amino acid residues are present in neither barnase nor binase. For some of the mutations, the improvement in proteolytic stability does not lead to a pronounced improvement in thermodynamic stability, indicating that the factors governing the proteolytic stability in some cases may be different from those governing the thermodynamic stability, e.g. propensity to local unfolding.The results obtained add important knowledge to a novel use of phage display technology for selection of thermodynamically stable proteins. Only by carefully establishing the parameters that can be adjusted, and recognising the influence this will have on the outcome of selection, will it be possible to realise the powerful technique of proteolytic selection.     

Comparative analysis of quantitative trait loci controlling glucosinolates,myrosinase and insect resistance in Arabidopsis thaliana

Evolutionary interactions among insect herbivores and plant chemical defenses have generated systems where plant compounds have opposing fitness consequences for host plants, depending on attack by various insect herbivores. This interplay complicates understanding of fitness costs and benefits of plant chemical defenses. We are studying the role of the glucosinolate-myrosinase chemical defense system in protecting Arabidopsis thaliana from specialist and generalist insect herbivory. We used two Arabidopsis recombinant inbred populations in which we had previously mapped QTL controlling variation in the glucosinolate-myrosinase system. In this study we mapped QTL controlling resistance to specialist (Plutella xylostella) and generalist (Trichoplusia ni) herbivores. We identified a number of QTL that are specific to one herbivore or the other, as well as a single QTL that controls resistance to both insects. Comparison of QTL for herbivory, glucosinolates, and myrosinase showed that T. ni herbivory is strongly deterred by higher glucosinolate levels, faster breakdown rates, and specific chemical structures. In contrast, P. xylostella herbivory is uncorrelated with variation in the glucosinolate-myrosinase system. This agrees with evolutionary theory stating that specialist insects may overcome host plant chemical defenses, whereas generalists will be sensitive to these same defenses.