Colour and in vitro quality attributes of walnuts from different growing conditions correlate with key precursors of primary and secondary metabolism.

Walnuts (Juglans regia L.) are well known for their flavour, nutritional and health properties. The light colour of walnuts is a quality attribute that leads to consumer preference. The aim of this study was to correlate attributes such as colour and antioxidant capacity with the precursors of primary and secondary metabolism. Two growing areas and four different colours of walnuts cv. Chandler from the central region of Chile were evaluated. Walnuts grown in the zone with Andes Mountains influence showed higher (p<0.05) sugar and unsaturated fatty acid contents, which could be attributed to lower minimum temperatures during seed filling. Extra light walnuts had higher (p<0.05) total phenolic compounds, antioxidant capacity and arbutin levels than amber walnuts. To the best of our knowledge, this is the first time that arbutin has been reported in walnuts and could provide the first insight into how enzymatic browning is prevented in the Chandler cultivar.

Enzymatic browning in avocado (Persea americana) revisited: History, advances, and future perspectives.

Considering nearly 80 years of research regarding one of the enzymes responsible for catalyzing the formation of pigments in higher animals, plants, fungi and bacteria, this review will focus on collecting and categorizing the existing information about polyphenol oxidase (PPO) in fruits, with particular emphasis on the information in relation to avocado, which is one of the hardiest species in terms of inactivation, has documented dual activity (EC 1.14.18.1/EC 1.10.3.1), and represents one of the oldest challenges for food science research and fruit processors. It is expected that this review will contribute to the further development of the field by highlighting the questions that have arisen during the characterization of PPO, the progress that has been made and the questions that remain today, in addition to new methodologies that are being applied to study this system. Holistic methodologies offer unexplored potential for advancing our understanding of the complex phenomena that govern PPO activity in fruits, because these methodologies will enable the characterization of this family of enzymes in all of its complexity. Subsequently, it will be possible to develop better techniques for controlling enzymatic browning in this valuable fruit.

Ligand-induced formation of transient dimers of mammalian 12/15-lipoxygenase: a key to allosteric behavior of this class of enzymes?

Mammalian lipoxygenases (LOXs) have been implicated in cellular defense response and are important for physiological homeostasis. Since their discovery, LOXs have been believed to function as monomeric enzymes that exhibit allosteric properties. In aqueous solutions, the rabbit 12/15-LOX is mainly present as hydrated monomer but changes in the local physiochemical environment suggested a monomer-dimer equilibrium. Because the allosteric character of the enzyme can hardly be explained using a single ligand binding-site model, we proposed that the binding of allosteric effectors may shift the monomer-dimer equilibrium toward dimer formation. To test this hypothesis, we explored the impact of an allosteric effector [13(S)-hydroxyoctadeca-9(Z),11(E)-dienoic acid] on the structural properties of rabbit 12/15-LOX by small-angle X-ray scattering. Our data indicate that the enzyme undergoes ligand-induced dimerization in aqueous solution, and molecular dynamics simulations suggested that LOX dimers may be stable in the presence of substrate fatty acids. These data provide direct structural evidence for the existence of LOX dimers, where two noncovalently linked enzyme molecules might work in unison and, therefore, such mode of association might be related to the allosteric character of 12/15-LOX. Introduction of negatively charged residues (W181E + H585E and L183E + L192E) at the intermonomer interface disturbs the hydrophobic dimer interaction of the wild-type LOX, and this structural alteration may lead to functional distortion of mutant enzymes.

Substrate binding to mammalian 15-lipoxygenase.

Lipoxygenases (LOs) are implicated in the regulation of metabolic processes and in several human diseases. Revealing their exact role is hindered by an incomplete understanding of their activity, including substrate specificity and substrate alignment in the active site. Recently, it has been proposed that the change in substrate specificity for arachidonic acid (AA) or linoleic acid (LA) could be part of an auto-regulatory mechanism related to cancer grow. Kinetic differences between reactions of 15-hLO with AA and LA have also led to the suggestion that the two substrates could present mechanistic differences. In the absence of a crystal structure for the substrate:15-LO complex, here we present an atomic-level study of catalytically competent binding modes for LA to rabbit 15-LO (15-rLO-1) and compare the results to our previous work on AA. Docking calculations, molecular dynamics simulations, re-docking and cross-docking calculations are all used to analyze the differences and similarities between the binding modes of the two substrates. Interestingly, LA seems to adapt more easily to the enzyme structure and differs from AA on some dynamical aspects that could introduce kinetic differences, as observed experimentally. Still, our study concludes that, despite the different chain lengths and number of insaturations between these two physiological substrates of 15-rLO-1, the enzyme seems to catalyze their hydroperoxidation by binding them with a common binding mode that leads to similar catalytically competent complexes.

Insights into the mechanism of binding of arachidonic acid to mammalian 15-lipoxygenases.

Mammalian 15-lipoxygenases (15-LOs) are key pharmaceutical targets under strong investigation because of their implication in atherosclerosis and cancer. Here, we present an atomic-level study of the binding modes of arachidonic acid (AA) to rabbit reticulocyte 15-LO, with a particular insight into the 15-LO:AA complexes consistent with known catalytic activity. We take into account both ligand and protein flexibility, by combining protein-ligand docking techniques and molecular dynamics simulations. We have also performed in silico mutagenesis. Our results are in agreement with previous mutagenesis data, in particular with the importance of Arg403 on AA binding. Interestingly, our results also reveal a central role of Arg403 in the conformational change of the alpha2-helix observed upon ligand binding. That induced-fit effect could give a possible framework for the molecular explanation of the known allosteric effect and questions the suitability of the inhibitor-bound crystal structure for accepting AA. Accounting for these dynamical considerations might improve the drug design process.