A series of pyrazolate-based dizinc(II) complexes has been synthesized and investigated as functional models for phosphoesterases, focusing on correlations between hydrolytic activity and molecular parameters of the bimetallic core. The Zn...Zn distance, the (bridging or nonbridging) position of the Zn-bound hydroxide nucleophile, and individual metal ion coordination numbers are controlled by the topology of the compartmental ligand scaffold. Species distributions of the various dizinc complexes in solution have been determined potentiometrically, and structures in the solid state have been elucidated by X-ray crystallography. The hydrolysis of bis(p-nitrophenyl)phosphate (BNPP) promoted by the dinuclear phosphoesterase model complexes has been investigated in DMSO/buffered water (1:1) at 50 degrees C as a function of complex concentration, substrate concentration, and pH. Coordination of the phosphodiester has been followed by ESI mass spectrometry, and bidentate binding could be verified crystallographically in two cases. Drastic differences in hydrolytic activity are observed and can be attributed to molecular properties. A significant decrease of the pK(a) of zinc-bound water is observed if the resulting hydroxide is involved in a strongly hydrogen-bonded intramolecular O(2)H(3) bridge, which can be even more pronounced than for a bridging hydroxide. Irrespective of the pK(a) of the Zn-bound water, a hydroxide in a bridging position evidently is a relatively poor nucleophile, while a nonbridging hydroxide position is more favorable for hydrolytic activity. Additionally, the metal array has to provide a sufficient number of coordination sites for activating both the substrate and the nucleophile, where phosphate diesters such as BNPP preferentially bind in a bidentate fashion, requiring a third site for water binding. Product inhibition of the active site by the liberated (p-nitrophenyl)phosphate is observed, and the product-inhibited complex could be characterized crystallographically. In that complex, the phosphate monoester is found to cap a rectangular array of four zinc ions composed of two bimetallic entities.

SummaryModifications to the composition of starch, the major component of wheat flour, can have a profound effect on the nutritional and technological characteristics of the flour's end products. The starch synthesized in the grain of conventional wheats (Triticum aestivum) is a 3:1 mixture of the two polysaccharides amylopectin and amylose. Altering the activity of certain key starch synthesis enzymes (GBSSI, SSIIa and SBEIIa) has succeeded in generating starches containing a different polysaccharide ratio. Here, mutagenesis, followed by a conventional marker‐assisted breeding exercise, has been used to generate three mutant lines that produce starch with an amylose contents of 0%, 46% and 79%. The direct and pleiotropic effects of the multiple mutation lines were identified at both the biochemical and molecular levels. Both the structure and composition of the starch were materially altered, changes which affected the functionality of the starch. An analysis of sugar and nonstarch polysaccharide content in the endosperm suggested an impact of the mutations on the carbon allocation process, suggesting the existence of cross‐talk between the starch and carbohydrate synthesis pathways.

Enzymatic reactions involving bilayer lipids occur in an environment with strict physical and topological constraints. The integral membrane enzyme PagP transfers a palmitoyl group from a phospholipid to lipid A in order to assist Escherichia coli in evading host immune defenses during infection. PagP measures the palmitoyl group with an internal hydrocarbon ruler that is formed in the interior of the eight-stranded antiparallel β barrel. The access and egress of the palmitoyl group is thought to take a lateral route from the bilayer phase to the barrel interior. Molecular dynamics, mutagenesis, and a 1.4 A crystal structure of PagP in an SDS / 2-methyl-2,4-pentanediol (MPD) cosolvent system reveal that phospholipid access occurs at the crenel present between strands F and G of PagP. In this way, the phospholipid head group can remain exposed to the cell exterior while the lipid acyl chain remains in a predominantly hydrophobic environment as it translocates to the protein interior.

The structure of the bis-intercalation complex of the depsipeptide antibiotic echinomycin with (CGTACG)2 has been redetermined at a higher resolution (1.4 A) and new high-resolution structures (1.1-1.5 A) are reported for the complexes of echinomycin with (GCGTACGC)2 (at both low and high ionic strengths) and (ACGTACGT)2. The structures show the expected Hoogsteen pairing for the base pairs flanking the intercalating chromophores on the outside and Watson-Crick pairing for both base pairs enclosed by the echinomycin. In the octamer complexes but not the hexamer complex, the echinomycin molecule, which would possess a molecular twofold axis were it not for the thioacetal bridge, shows twofold disorder. In all the structures the stacking of the base pairs and chromophores is extended by intermolecular stacking. The structures provide more precise details of the hydrogen bonding and other interactions between the bis-intercalating antibiotics and the duplex DNA than were previously available.