Crystal structure of bifunctional aldos-2-ulose dehydratase/isomerase from Phanerochaete chrysosporium with the reaction intermediate ascopyrone M

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn²⁺ and Mg²⁺ located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn²⁺-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn²⁺ in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn²⁺ acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.     

The significance of host-fungus combinations in ectomycorrhizal symbioses for the chemical quality of willow foliage

The majority of plants interacts with mycorrhizal fungi, which predominantly provide mutual benefits, but also costs. We tested the hypothesis that specific combinations of host plants (four commercial varieties of Salix spp.) and ectomycorrhizal partners (species of Laccaria, Paxillus, Tricholoma and Hebeloma) differ in their effects on the host foliar chemistry. Twenty specific host - mycorrhiza combinations were pot-grown outdoors under low-N conditions. Foliar concentrations of total phenolics and salicylic acid were decreased by mycorrhizas in S. schwerinii x S. viminalis and S. x dasyclados genotypes, but increased in S. viminalis. Mycorrhiza effects on host biomass production were positive, indifferent or negative, depending on the specific host—mycorrhiza combination. The host plant genotype influenced mainly the direction of mycorrhizal effects on foliar chemistry, whereas the magnitude of mycorrhizal effects varied mostly among the fungal genotypes. The results are relevant with respect to the possible interactions between mycorrhizas, plants and leaf herbivores.     

Desaturases: Emerging Models for Understanding Functional Diversification of Diiron-containing Enzymes

Desaturases and related enzymes perform O₂-dependent dehydrogenations initiated at unactivated C-H groups with the use of a diiron active site. Determination of the long-sought oxidized desaturase crystal structure facilitated structural comparison of the active sites of disparate diiron enzymes. Experiments on the castor desaturase are discussed that provide experimental support for a hypothesized ancestral oxidase enzyme in the context of the evolution of the diiron enzyme diverse functionality. We also summarize recent analysis of a castor mutant desaturase that provides valuable insights into the relationship of proposed substrate-binding modes with respect to a range of catalytic outcomes.Desaturase enzymes perform dehydrogenation reactions that result in the introduction of double bonds into fatty acids that are initiated by the energy-demanding abstraction of a hydrogen from a methylene group (1–3). To achieve this, desaturase enzymes recruit and activate molecular oxygen with the use of an active-site diiron cluster (4). The diiron center is common to a variety of proteins, including methane monooxygenase, ribonucleotide reductase, rubrerythrins, and a variety of oxidase enzymes (5). Valuable insights regarding the tuning of diiron centers with respect to diverse chemical reactivity (6) have been made via comparisons of the diiron centers of diiron-containing enzymes (7); however, differences in amino acid sequence, multiple protein-protein interactions, and reaction outcomes complicate the analysis. The study of fatty-acid desaturases and related enzymes presents a unique opportunity for performing enzyme structure-function studies because relatively close homologs perform diverse reactions on similar substrates (8, 9).Desaturase enzymes have evolved independently twice (10); the acyl-ACP² desaturases are soluble enzymes found in the plastids of higher plants, whereas the more widespread class of integral membrane desaturases is found in endomembrane systems in prokaryotes and eukaryotes (9). In addition to forming distinct homology groups, their diiron centers possess distinct primary ligation spheres (11). The availability of crystal structures for acyl-ACP desaturases (12) makes this system amenable to detailed structure-function studies. Crystal structures are available for the 18:0 Δ⁹-desaturase³ (12, 13) from Ricinus communis (castor) and a bifunctional desaturase from Hedera helix (ivy) (14, 15). These desaturases are homodimeric proteins, with each monomer folded into a compact single domain composed of nine helices. The diiron active site of these enzymes is buried within a core four-helix bundle and is positioned alongside a deep, bent, narrow hydrophobic cavity in which the substrate is bound during catalysis. It is a textbook example of a lock-and-key type of binding site in which the bound fatty acid moiety is poised for formation of the cis-fatty acid product.Nobel Laureate Konrad Bloch observed, “The stereospecific removal of hydrogen in the formation of oleate, although predictable on principle grounds would seem to approach the limits of the discriminatory power of enzymes” (16). Bloch''s statement underscores that desaturase enzymes perform highly regio- and stereo-selective reactions on long-chain fatty acids composed of essentially equivalent methylene chains that lack distinguishing landmarks close to the site of desaturation. We will review structural features of the diiron active site of the acyl-ACP desaturases in the context of those of other diiron enzymes, discuss recent insights into the evolution of acyl-ACP desaturases, and summarize recent discoveries relating to the evolution of selectivity and functional diversity within desaturase enzyme families.     

Lipid droplets interact with mitochondria using SNAP23

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.     

Study of antimicrobial activity amongLactobacillus helveticus strains using three different assays

The antimicrobial activity of 18Lactobacillus helveticus strains as well as one control strain, the bacteriocin producingLactobacillus helveticus 481, was tested with three different inhibition assays. FourLactobacillus helveticus strains had antimicrobial activity against seven otherLactobacillus helveticus strains and twoLactobacillus delbrueckil strains while the remaining sevenLactobacillus helveticus strains were indifferent. Inhibition was also observed againstLactococcus andLeuconostoc species, due to production of organic acids or hydrogen peroxide. The strains with the highest antimicrobial activity produced a heat sensitive proteinacious bacteriocin with a narrow species activity spectrum against only thermophilicLactobacillus strains.     

Synthesis and SAR of potent inhibitors of the Hepatitis C virus NS3/4A protease: Exploration of P2 quinazoline substituents

Novel NS3/4A protease inhibitors comprising quinazoline derivatives as P2 substituent were synthesized. High potency inhibitors displaying advantageous PK properties have been obtained through the optimization of quinazoline P2 substituents in three series exhibiting macrocyclic P2 cyclopentane dicarboxylic acid and P2 proline urea motifs. For the quinazoline moiety it was found that 8-methyl substitution in the P2 cyclopentane dicarboxylic acid series improved on the metabolic stability in human liver microsomes. By comparison, the proline urea series displayed advantageous Caco-2 permeability over the cyclopentane series. Pharmacokinetic properties in vivo were assessed in rat on selected compounds, where excellent exposure and liver-to-plasma ratios were demonstrated for a member of the 14-membered quinazoline substituted P2 proline urea series.     

Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination

In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.     

Anaerobic energy expenditure and mechanical efficiency during exhaustive leg press exercise

Information about anaerobic energy production and mechanical efficiency that occurs over time during short-lasting maximal exercise is scarce and controversial. Bilateral leg press is an interesting muscle contraction model to estimate anaerobic energy production and mechanical efficiency during maximal exercise because it largely differs from the models used until now. This study examined the changes in muscle metabolite concentration and power output production during the first and the second half of a set of 10 repetitions to failure (10RM) of bilateral leg press exercise. On two separate days, muscle biopsies were obtained from vastus lateralis prior and immediately after a set of 5 or a set of 10 repetitions. During the second set of 5 repetitions, mean power production decreased by 19% and the average ATP utilisation accounted for by phosphagen decreased from 54% to 19%, whereas ATP utilisation from anaerobic glycolysis increased from 46 to 81%. Changes in contraction time and power output were correlated to the changes in muscle Phosphocreatine (PCr; r = −0.76; P<0.01) and lactate (r = −0.91; P<0.01), respectively, and were accompanied by parallel decreases (P<0.01-0.05) in muscle energy charge (0.6%), muscle ATP/ADP (8%) and ATP/AMP (19%) ratios, as well as by increases in ADP content (7%). The estimated average rate of ATP utilisation from anaerobic sources during the final 5 repetitions fell to 83% whereas total anaerobic ATP production increased by 9% due to a 30% longer average duration of exercise (18.4±4.0 vs 14.2±2.1 s). These data indicate that during a set of 10RM of bilateral leg press exercise there is a decrease in power output which is associated with a decrease in the contribution of PCr and/or an increase in muscle lactate. The higher energy cost per repetition during the second 5 repetitions is suggestive of decreased mechanical efficiency.