Posttranslational modifications of histones regulate chromatin structure and gene expression. Histone demethylases, members of a newly emerging transcription-factor family, remove methyl groups from the lysine residues of the histone tails and thereby regulate the transcriptional activity of target genes. JmjC-domain-containing proteins have been predicted to be demethylases. For example, the JmjC-containing protein JMJD2A has been characterized as a H3-K9me3- and H3-K36me3-specific demethylase. Here, structures of the catalytic-core domain of JMJD2A with and without alpha-ketoglutarate in the presence of Fe2+ have been determined by X-ray crystallography. The structure of the core domain, consisting of the JmjN domain, the JmjC domain, the C-terminal domain, and a zinc-finger motif, revealed the unique elements that form a potential substrate binding pocket. Sited-directed mutagenesis in conjunction with demethylase activity assays allowed us to propose a molecular model for substrate selection by the JMJD2 histone demethylase family.

Fucoxanthin, one of the main marine carotenoids, is abundant in macro- and microalgae. Here, fucoxanthin was isolated and structurally identified as the major carotenoid in the diatom Phaeodactylum tricornutum through chromatographic and spectroscopic methods, such as liquid chromatography-positive-ion atmospheric pressure chemical ionization/mass spectroscopy and nuclear magnetic resonance. This pigment was quantified by reverse-phase high-performance liquid chromatography, and a number of extraction procedures were assessed to investigate the effect of solvent type, extraction time, temperature, and extraction method (maceration, ultrasound-assisted extraction, Soxhlet extraction, and pressurized liquid extraction). Among the investigated solvents, ethanol provided the best fucoxanthin extraction yield (15.71 mg/g freeze-dried sample weight). Fucoxanthin content in the extracts produced by the different methods was quite constant (15.42-16.51 mg/g freeze-dried sample weight) but increased steeply based on the percentage of ethanol in water, emphasizing the importance of ethanol in the extraction. The results indicate that P. tricornutum is a rich source of fucoxanthin (at least ten times more abundant than that in macroalgae) that is easily extracted with ethanol, suggesting potential applications in human and animal food, health, and cosmetics.

The sarA locus in Staphylococcus aureus controls the expression of many virulence genes. The sarA regulatory molecule, SarA, is a 14.7-kDa protein (124 residues) that binds to the promoter region of target genes. Here we report the 2.6 Å-resolution x-ray crystal structure of the dimeric winged helix SarA protein, which differs from the published SarA structure dramatically. In the crystal packing, multiple dimers of SarA form a scaffold, possibly via divalent cations. Mutations of individual residues within the DNAbinding helix-turn-helix and the winged region as well as within the metal-binding pocket implicate basic residues R84 and R90 within the winged region to be critical in DNA binding, whereas acidic residues D88 and E89 (wing), D8 and E11 (metal-binding pocket), and cysteine 9 are essential for SarA function. These data suggest that the winged region of the winged helix protein participates in DNA binding and activation, whereas the putative divalent cation binding pocket is only involved in gene function.

JMJD6 is a Jumonji C domain-containing hydroxylase. JMJD6 binds α-ketoglutarate and iron and has been characterized as either a histone arginine demethylase or U2AF65 lysyl hydroxylase. Here, we describe the structures of JMJD6 with and without α-ketoglutarate, which revealed a novel substrate binding groove and two positively charged surfaces. The structures also contain a stack of aromatic residues located near the active center. The side chain of one residue within this stack assumed different conformations in the two structures. Interestingly, JMJD6 bound efficiently to single-stranded RNA, but not to single-stranded DNA, doublestranded RNA, or double-stranded DNA. These structural features and truncation analysis of JMJD6 suggest that JMJD6 may bind and modify single-stand RNA rather than the previously reported peptide substrates.RNA binding proteins | RNA modification | RNA splicing J MJD6 was first characterized as a receptor for phosphatidylserine (PSR), which facilitates the phagocytosis of dead and dying cells by macrophages and fibroblasts (1). Targeted deletion of gene encoding PSR in mice and morpholino knock-downs of PSR in zebrafish resulted in embryonic lethality, with severe defects in hematopoiesis and aberrant development of eye, brain, and heart (2-5). In contrast, knock-down of PSR expression in Caenorhabditis elegans produced only a mild phenotype (5). Somewhat surprisingly, sequence analysis suggested that JMJD6 contains a Jumonji C (JMJC) domain, which places it within a highly conserved, cupin fold-containing enzyme family (6-8). Further analysis demonstrated that the protein is localized specifically in the nucleus (7-9). Despite the significant effects of JMJD6 deficiency, knockout mice engulfed apoptotic cells normally (9). Based on these studies and additional sequence analysis, the protein was recategorized as an α-ketoglutarateand Fe 2þ -dependent hydroxylase and was named JMJD6 (10).Recent studies demonstrated that most JMJC domain-containing proteins function as histone demethylases by specifically acting on lysine residues in histone tails (11)(12)(13)(14). For example, the specific interactions between enzymes from the JMJD2 subfamily and methylated peptides have been structurally characterized (15-18). Interestingly, JMJD6 was reported to demethylate arginine residues in histone tails (10). Several laboratories including ours, however, have been unable to reproduce these results. In other studies, JMJD6 was identified as a lysine hydroxylase that specifically recognizes the protein tail of U2AF65, a mediator of RNA splicing (19).To resolve the disparate results and further elucidate the structure and functions of JMJD6, we determined X-ray crystallographic structures of the protein with and without α-ketoglutarate. To obtain these structures, JMJD6 was cocrystallized with a Fab fragment derived from a JMJD6-specific hamster monoclonal antibody. Intriguingly, the structure of JMJD6 is dramatically different from known structures of other JMJC domain superfamily proteins includ...