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The SAGA (Spt-Ada-Gcn5 acetyltransferase) complex is an important chromatin modifying complex that can both acetylate and deubiquitinate histones. Sgf29 is a novel component of the SAGA complex. Here, we report the crystal structures of the tandem Tudor domains of Saccharomyces cerevisiae and human Sgf29 and their complexes with H3K4me2 and H3K4me3 peptides, respectively, and show that Sgf29 selectively binds H3K4me2/3 marks. Our crystal structures reveal that Sgf29 harbours unique tandem Tudor domains in its C-terminus. The tandem Tudor domains in Sgf29 tightly pack against each other face-to-face with each Tudor domain harbouring a negatively charged pocket accommodating the first residue alanine and methylated K4 residue of histone H3, respectively. The H3A1 and K4me3 binding pockets and the limited binding cleft length between these two binding pockets are the structural determinants in conferring the ability of Sgf29 to selectively recognize H3K4me2/3. Our in vitro and in vivo functional assays show that Sgf29 recognizes methylated H3K4 to recruit the SAGA complex to its targets sites and mediates histone H3 acetylation, underscoring the importance of Sgf29 in gene regulation. [ABSTRACT FROM AUTHOR]

Ephrin receptor tyrosine kinase A3 (EphA3, EC 2.7.10.1) is a member of a unique branch of the kinome in which downstream signaling occurs in both ligand- and receptor-expressing cells. Consequently, the ephrins and ephrin receptor tyrosine kinases often mediate processes involving cell–cell contact, including cellular adhesion or repulsion, developmental remodeling and neuronal mapping. The receptor is also frequently overexpressed in invasive cancers, including breast, small-cell lung and gastrointestinal cancers. However, little is known about direct substrates of EphA3 kinase and no chemical probes are available. Using a library approach, we found a short peptide sequence that is a good substrate for EphA3 and is suitable for co-crystallization studies. Complex structures show multiple contacts between kinase and substrates; in particular, two residues undergo conformational changes and by mutation are found to be important for substrate binding and turnover. In addition, a difference in catalytic efficiency between EPH kinase family members is observed. These results provide insight into the mechanism of substrate binding to these developmentally integral enzymes. [ABSTRACT FROM AUTHOR]

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Histone methylation at specific lysine residues brings about various downstream events that are mediated by different effector proteins. The WD40 domain of WDR5 represents a new class of histone methyl-lysine recognition domains that is important for recruiting H3K4 methyltransferases to K4-dimethylated histone H3 tail as well as for global and gene-specific K4 trimethylation. Here we report the crystal structures of full-length WDR5, WDR5Δ23 and its complexes with unmodified, mono-, di- and trimethylated histone H3K4 peptides. The structures reveal that WDR5 is able to bind all of these histone H3 peptides, but only H3K4me2 peptide forms extra interactions with WDR5 by use of both water-mediated hydrogen bonding and the altered hydrophilicity of the modified lysine 4. We propose a mechanism for the involvement of WDR5 in binding and presenting histone H3K4 for further methylation as a component of MLL complexes. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenases are general detoxifying enzymes, but there are also isoenzymes that are involved in specific metabolic pathways in different organisms. Two of these enzymes are Escherichia coli lactaldehyde (ALD) and phenylacetaldehyde dehydrogenases (PAD), which participate in the metabolism of fucose and phenylalanine, respectively. These isozymes share some properties with the better characterized mammalian enzymes but have kinetic properties that are unique. It was possible to thread the sequences into the known ones for the mammalian isozymes to better understand some structural differences. Both isozymes were homotetramers, but PAD used both NAD+ and NADP+ but with a clear preference for NAD, while ALD used only NAD+. The rate-limiting step for PAD was hydride transfer as indicated by the primary isotopic effect and the absence of a pre-steady-state burst, something not previously found for tetrameric enzymes from other organisms where the rate-limiting step is related to both deacylation and coenzyme dissociation. In contrast, ALD had a pre-steady-state burst indicating that the rate-limiting step was located after the NADH formation, but the rate-limiting step was a combination of deacylation and coenzyme dissociation. Both enzymes possessed esterase activity that was stimulated by NADH; NAD+ stimulated the esterase activity of PAD but not of ALD. Finding enzymes that structurally are similar to the well-characterized mammalian enzymes but have a different rate-limiting step might serve as models to allow us to determine what regulates the rate-limiting step. [ABSTRACT FROM AUTHOR]

To gain a better insight into the alterations of brain function after chronic ethanol, we measured the release of various neurotransmitters from nerve terminals of cortex and hippocampus isolated from rats chronically fed with ethanol. The K[SUP+]-evoked release of [SUP3]H]acetylcholine (ACh), [ [SUP3]H]dopamine (DA), [SUP3]H]glutamate (Glu) and [SUP3]H]noradrenaline (NA) was determined in superfused synaptosomes of brain cortex and hippocampus from rats exposed to the Lieber-DeCarli alcohol liquid diet for 5 weeks. In cortical synaptosomes, chronic ethanol administration did not affect the release of ACh and of DA, while significantly decreasing the release of Glu and NA. The endogenous levels of NA, DA and their metabolites were unchanged. In hippocampal synaptosomes the only effect of chronic alcohol was an increased release of Glu. It can be concluded that at presynaptic level chronic ethanol alters brain neurotransmitter systems selectively. Glutamatergic and noradrenergic nerve terminals from cortex are more vulnerable than those from hippocampus. [ABSTRACT FROM AUTHOR]

Human stomach mucosa contains a characteristic alcohol dehydrogenase (ADH) enzyme, σσADH. Its cDNA has been cloned from a human stomach library and sequenced. The deduced amino acid sequence shows 59-70% identities with the other human ADH classes, demonstrating that the stomach enzyme represents a distinct structure, constituting class IV, coded by a separate gene. ADH7. The amino acid identity with the rat stomach class IV ADH is 88%, which is intermediate between constant and variable dehydrogenases. This value reflects higher conservation than for the classical liver enzymes of class I, compatible with a separate functional significance of the class IV enzyme. Its enzymic features can be correlated with its structural characteristics. The residues lining the substrate-binding cleft are bulky and hydmphobic, similar to those of the class I enzyme: this explains the similar specificity of both classes, compatible with the origin of class IV from class I. Position 47 has Arg, in contrast to Gly in the rat class IV enzyme, bm this Arg is still associated with an extremely high activity (kcat = 1510 min-1) and weak coenzyme binding (KiaNAD+ = 1.6 mM). Thus, the strong interaction with coenzyme imposed by Arg47 in class I is probably compensated for in crass IV by changes that may negatively affect coenzyme binding: Glu230, His271, Ash260, Asn261, Asn363. The still higher activity and weaker coenzyme binding of rat class IV (kcat = 2600 min-1, KiaNAD = 4 mM) can be correlated to the exchanges to Gly47, Gln230 and Tyr363. An important change at position 294, with Val in human and Ala in rat class IV, is probably responsible for the dramatic difference in Km values for ethanol between human (37 mM) and rat (2.4 M) class IV enzymes. [ABSTRACT FROM AUTHOR]