No abstract

Epigenetic indexing of chromatin domains by histone lysine methylation requires the balanced coordination of methyltransferase and demethylase activities. Here, we show that SU(VAR)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. SU(VAR)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. Our data indicate an early developmental function for the SU(VAR)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which SU(VAR)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation.

Cells contain numerous enzymes that use molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein, which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D freeenergy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics.free energy ͉ lipid peroxidation ͉ oxygen channel ͉ oxygen diffusion ͉ oxygenases

Multiple lipoxygenase sequence alignments and structural modeling of the enzyme͞substrate interaction of the cucumber lipid body lipoxygenase suggested histidine 608 as the primary determinant of positional specificity. Replacement of this amino acid by a less-space-filling valine altered the positional specificity of this linoleate 13-lipoxygenase in favor of 9-lipoxygenation. These alterations may be explained by the fact that H608V mutation may demask the positively charged guanidino group of R758, which, in turn, may force an inverse head-to-tail orientation of the fatty acid substrate. The R758L؉H608V double mutant exhibited a strongly reduced reaction rate and a random positional specificity. Trilinolein, which lacks free carboxylic groups, was oxygenated to the corresponding (13S)-hydro(pero)xy derivatives by both the wild-type enzyme and the linoleate 9-lipoxygenating H608V mutant. These data indicate the complete conversion of a linoleate 13-lipoxygenase to a 9-lipoxygenating species by a single point mutation. It is hypothesized that H608V exchange may alter the orientation of the substrate at the active site and͞or its steric configuration in such a way that a stereospecific dioxygen insertion at C-9 may exclusively take place.Lipoxgenases (LOXs; linoleate:oxygen oxidoreductase; EC 1.13.11.12) are widely distributed in the plant and animal kingdom (1, 2). They constitute a family of nonheme ironcontaining dioxygenases that catalyze the regio-and stereoselective dioxygenation of polyenoic fatty acids forming hydroperoxy derivatives (3). In mammals, LOXs are classified according to their positional specificity of arachidonic acid oxygenation (2, 4). Because arachidonic acid either is not present in higher plants or is a minor constituent of cellular lipids, plant LOXs are classified into 9-and 13-LOXs with respect to their positional specificity of linoleic acid (LA) oxygenation (5). Recently, a more comprehensive classification of plant LOXs has been proposed based on the comparison of their primary structures (6).The positional specificity of LOXs is a result of two catalytic processes. (i) Regio-and stereospecific hydrogen removal; with substrate fatty acids containing several doubly allylic methylenes such as linolenic acid, arachidonic acid, or eicosapentaenoic acid hydrogen abstraction from two, three, or four doubly allylic methylenes, respectively, is possible. (ii) Regio-and stereospecific oxygen insertion: when hydrogen is abstracted from a certain doubly allylic methylene, molecular oxygen can be introduced either at the [ϩ2] or at the [Ϫ2] position (Fig. 1). Thus, a fatty acid containing three doubly allylic methylenes such as arachidonic acid can be oxygenated by a LOX to six regioisomeric hydroperoxy derivatives (HPETEs), namely 15-and 11-HPETE (originating from C-13 hydrogen removal), 12-and 8-HPETE (C-10 hydrogen removal), and 9-and 5-HPETE (C-7 hydrogen removal). Experiments on mammalian 12-and 15-LOXs indicated that the site of hydrogen abstraction can be altered when critical ...

The nomenclature of lipoxygenases (LOXs) is partly based on the positional specificity of arachidonic acid oxygenation, but there is no unifying concept explaining the mechanistic basis of this enzyme property. According to the triad model, Phe-353, Ile-418, and Ile-593 of the rabbit 12/15-LOX form the bottom of the substrate-binding pocket, and introduction of less spacefilling residues at either of these positions favors arachidonic acid 12-lipoxygenation. The present study was aimed at exploring the validity of the triad concept for two novel primate 12/15-LOX (Macaca mulatta and Pongo pygmaeus) and for five known members of the mammalian LOX family (human 12/15-LOX, mouse 12/15-LOX, human 15-LOX2, human platelet type 12-LOX, and mouse (12R)-LOX). The enzymes were expressed as N-terminal His tag fusion proteins in E. coli, the potential sequence determinants were mutated, and the specificity of arachidonic acid oxygenation was quantified. Taken together, our data indicate that the triad concept explains the positional specificity of all 12/15-LOXs tested (rabbit, human, M. mulatta, P. pygmaeus, and mouse). For the new enzymes of M. mulatta and P. pygmaeus, the concept had predictive value because the positional specificity predicted on the basis of the amino acid sequence was confirmed experimentally. The specificity of the platelet 12-LOX was partly explained by the triad hypothesis, but the concept was not applicable for 15-LOX2 and (12R)-LOX.