Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal ␣-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.3D structure ͉ ammonia tunnel ͉ glutamine amidotransferase ͉ oxyanion ͉ vitamin B6

The process of mononucleosome assembly mediated by histone chaperone NAP1 was investigated using DNA fragments 146 and 207 bp in length containing the Lytechinus variegatus 5 S rDNA nucleosome positioning sequence. A quantitative description was derived using gel electrophoresis and fluorescent anisotropy data. First, NAP1-bound H3⅐H4 was released forming a DNA-histone tetramer complex with a time constant of k 1 ‫؍‬ (2.5 ؎ 0.7) ⅐ 10 4 M ؊1 s ؊1 .The tetrasome was converted quickly (k 2 ‫؍‬ (4.1 ؎ 3.5) ⅐ 10 5 M ؊1 s ؊1 ), by the addition of a single H2A⅐H2B dimer, into a "hexasome,"i.e. a nucleosome lacking one H2A⅐H2B dimer. From this intermediate a nucleosome was formed by the addition of a second H2A⅐H2B dimer with an average rate constant k 3 ‫؍‬ (6.6 ؎ 1.4) ⅐ 10 3 M ؊1 s ؊1 . For the back-reaction, significant differences were observed between the 146-and 207-bp DNA upon substitution of the canonical H2A histone with H2A.Z. The distinct nucleosome/ hexasome ratios were reflected in the corresponding equilibrium dissociation constants and revealed some differences in nucleosome stability. In a fourth reaction, NAP1 mediated the binding of linker histone H1 to the nucleosome, completing the chromatosome structure with k 4 ‫؍‬ (7.7 ؎ 3.7) ⅐ 10 3 M ؊1 s ؊1 . The activity of the chromatin remodeling complex ACF did not increase the kinetics of the mononucleosome assembly process.The repeating building block of chromatin is the nucleosome, a nucleoprotein complex consisting of two each of histones H2A, H2B, H3, and H4 wrapped around 146/147 bp of DNA (1). Several studies have focused on the mechanism by which these entities are assembled and how a defined chromatin structure is established (2-10). In vivo chromatin assembly is mostly coupled to DNA replication (11), but recent investigations have pointed out the importance of replicationindependent deposition of variant histones such as H2A.Z and H3.3. This process appears to be relevant in the formation of chromatin with differential transcriptional activity (12-16). Assembly of nucleosomes seems to be closely linked to the activity of ATP-dependent chromatin remodeling machineries, required for the formation of evenly spaced nucleosome arrays, which are characteristic for the native chromatin conformation (17).Simple mixing of histones and DNA leads mainly to large, insoluble aggregates (18,19). Accordingly, the transfer of histones to DNA in the cell is carried out by histone chaperones such as the heterotrimeric chromatin assembly factor 1 (CAF1) (20), N1/N2 (21, 22), nucleoplasmin (23-25), HIRA (26,27), and nucleosome assembly protein 1 (NAP1) 2 (28, 29). Per definition, all share the capability of binding histones and releasing them to DNA or other targets, but they differ with respect to in vivo functions and the preferred histone interaction partner. The histone chaperone NAP1 studied here is involved in the shuttling of newly synthesized histone H2A⅐H2B dimers from the cytoplasm to the nucleus and the deposition of histones onto the DNA (3, 17, 30). In a...

The ATPase ISWI is the molecular motor of several nucleosome remodeling complexes including ACF. We analyzed the ACF-nucleosome interactions and determined the characteristics of ACF-dependent nucleosome remodeling. In contrast to ISWI, ACF interacts symmetrically with DNA entry sites of the nucleosome. Two-color fluorescence cross-correlation spectroscopy measurements show that ACF can bind four DNA duplexes simultaneously in a complex that contains two Acf1 and ISWI molecules. Using bead-bound nucleosomal substrates, nucleosome movement by mechanisms involving DNA twisting was excluded. Furthermore, an ACF-dependent local detachment of DNA from the nucleosome was demonstrated in a novel assay based on the preferred intercalation of ethidium bromide to free DNA. The findings suggest a loop recapture mechanism in which ACF introduces a DNA loop at the nucleosomal entry site that propagates over the histone octamer surface and leads to nucleosome repositioning.