A key step in many chromatin-related processes is the recognition of histone post-translational modifications by effector modules such as bromodomains and chromo-like domains of the Royal family. Whereas effector-mediated recognition of single post-translational modifications is well characterized, how the cell achieves combinatorial readout of histones bearing multiple modifications is poorly understood. One mechanism involves multivalent binding by linked effector modules. For example, the tandem bromodomains of human TATA-binding protein-associated factor-1 (TAF1) bind better to a diacetylated histone H4 tail than to monoacetylated tails, a cooperative effect attributed to each bromodomain engaging one acetyl-lysine mark. Here we report a distinct mechanism of combinatorial readout for the mouse TAF1 homologue Brdt, a testis-specific member of the BET protein family. Brdt associates with hyperacetylated histone H4 (ref. 7) and is implicated in the marked chromatin remodelling that follows histone hyperacetylation during spermiogenesis, the stage of spermatogenesis in which post-meiotic germ cells mature into fully differentiated sperm. Notably, we find that a single bromodomain (BD1) of Brdt is responsible for selectively recognizing histone H4 tails bearing two or more acetylation marks. The crystal structure of BD1 bound to a diacetylated H4 tail shows how two acetyl-lysine residues cooperate to interact with one binding pocket. Structure-based mutagenesis that reduces the selectivity of BD1 towards diacetylated tails destabilizes the association of Brdt with acetylated chromatin in vivo. Structural analysis suggests that other chromatin-associated proteins may be capable of a similar mode of ligand recognition, including yeast Bdf1, human TAF1 and human CBP/p300 (also known as CREBBP and EP300, respectively). Our findings describe a new mechanism for the combinatorial readout of histone modifications in which a single effector module engages two marks on a histone tail as a composite binding epitope.

The 'Synergistes' group, which apparently represents an as yet unnamed division of the bacteria, was explored in 93 anaerobic environments (guts, soils, digestors, etc.). From 16S rDNA gene-targeted polymerase chain reaction (PCR) assays, this group appeared to be present in 90% of the anaerobic microbial ecosystems analysed. The phylogeny of 103 16S rDNA sequences from 30 ecosystems showed a strong link between 16S rDNA sequences and given ecosystems. 'Synergistes' 16S rDNA sequences from animal sources (termites, guinea pigs, pigs, birds, etc.) formed clustered phylogenetical groups. 'Synergistes' groups were also associated either with anaerobic digestors and soils or with thermophilic conditions. Sequences available from the DNA database were consistent with the results. These results show the wide diversity of the 'Synergistes' division as well as the specific ecological niche of each 16S rDNA sequences.

Plant ␤-glucosidases play a crucial role in defense against pests. They cleave, with variable specificity, ␤-glucosides to release toxic aglycone moieties. The Sorghum bicolor ␤-glucosidase isoenzyme Dhr1 has a strict specificity for its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-␤-D-glucoside), whereas its close homolog, the maize ␤-glucosidase isoenzyme Glu1, which shares 72% sequence identity, hydrolyzes a broad spectrum of substrates in addition to its natural substrate 2-O-␤-Dglucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one. Structural data from enzyme⅐substrate complexes of Dhr1 show that the mode of aglycone binding differs from that previously observed in the homologous maize enzyme. Specifically, the data suggest that Asn Carbohydrates and their glycoconjugates are one of the most diverse groups of organic molecules in the biosphere. The selective cleavage of glycosidic bonds is crucial in a variety of fundamental biological processes for all living organisms. The large (and growing) number of glycoside hydrolase families reflects this diversity of substrates and the need for selective cleavage of the glycosidic bond. Ninety-one glycoside hydrolase families are currently available on the continuously updated CAZY web server (1).1 ␤-Glucosidases constitute a major group in glycoside hydrolase families 1 and 3 and hydrolyze either O-linked ␤-glycosidic bonds (␤-D-glucoside glucohydrolase, EC 3.2.1.21) or S-linked ␤-glycosidic bonds (myrosinase or ␤-Dthioglucoside glucohydrolase, EC 3.2.3.1). More precisely, enzymes of glycoside hydrolase family 1 hydrolyze substrates of the type G-O/S-X, where G indicates the glycosyl residue and X can be either another glycosyl residue or a non-glycosyl aglycone group. In higher plants, the major functions of ␤-glucosidases are defense against pests with the release of bitter or toxic aglycones and their breakdown products (2, 3), phytohormone activation (4, 5), lignification (6), and cell wall catabolism (7). The nature of the aglycone moiety of substrates is believed to be critical for the specificity and physiological functions of these enzymes.Because plants possess a large number of ␤-glucosidases (most of the 48 genes identified as putative ␤-glucosidase genes in Arabidopsis thaliana do not have a known function), the understanding of the mechanism of substrate specificity is important for accurately predicting the diverse physiological functions of these proteins. The roles of the 2 catalytic glutamates (8, 9) included in the TFNEP and Y(I/V)TENG peptide motifs of ␤-glucosidases as the general acid/base catalyst and the nucleophile, respectively (10), are now well understood. These residues are 5.5-6 Å apart within the active-site pocket on opposite sides of the glycosidic bond (8,11) and are required in the two steps of the substrate hydrolysis that results in retention of the anomeric conformation of C-1 at the point of cleavage. The conformational change in the glucose moiety prior to nucleophilic attack and the determinants of binding and d...

Plant ␤-glucosidases display varying substrate specificities. The maize ␤-glucosidase isozyme Glu1 (ZmGlu1) hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOA-Glc (2-O-␤-D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one), whereas the sorghum ␤-glucosidase isozyme Dhr1 ( , and Phe 473 of Glu1 by Dhr1 counterparts. The effects of mutations on enzyme activity and substrate specificity were studied using both natural and artificial substrates. The simple mutant replacing Phe 198 by a valine had the most drastic effect on activity, because the capacity of this enzyme to hydrolyze ␤-glucosides was almost completely abolished. The analysis of this mutation was completed by a structural study of the double mutant ZmGlu1-E191D,F198V in complex with the natural substrate. The structure reveals that the single mutation F198V causes a cascade of conformational changes, which are unpredictable by standard molecular modeling techniques. Some other mutations led to drastic effects: replacing Asp 261 by an asparagine decreases the catalytic efficiency of this simple mutant by 75% although replacing Tyr 473 by a phenylalanine increase its efficiency by 300% and also provides a new substrate specificity by hydrolyzing dhurrin.