Amylopectin Chain Length Dynamics and Activity Signatures of Key Carbon Metabolic Enzymes Highlight Early Maturation as Culprit for Yield Reduction of Barley Endosperm Starch after Heat Stress.

Abiotic environmental stresses have a negative impact on the yield and quality of crops. Understanding these stresses is an essential enabler for mitigating breeding strategies and it becomes more important as the frequency of extreme weather conditions increases due to climate change. This study analyses the response of barley (Hordeum vulgare L.) to a heat wave during grain filling in three distinct stages: the heat wave itself, the return to a normal temperature regime, and the process of maturation and desiccation. The properties and structure of the starch produced were followed throughout the maturational stages. Furthermore, the key enzymes involved in the carbohydrate supply to the grain were monitored. We observed differences in starch structure with well-separated effects because of heat stress and during senescence. Heat stress produced marked effects on sucrolytic enzymes in source and sink tissues. Early cessation of plant development as an indirect consequence of the heat wave was identified as the major contributor to final yield loss from the stress, highlighting the importance for functional stay-green traits for the development of heat-resistant cereals.

Structure of a dimeric fungal α-type carbonic anhydrase.

The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the ß class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.

PagP crystallized from SDS/cosolvent reveals the route for phospholipid access to the hydrocarbon ruler.

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 ß 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.

Insights into strand exchange in BTB domain dimers from the crystal structures of FAZF and Miz1.

The BTB domain is a widely distributed protein-protein interaction motif that is often found at the N-terminus of zinc finger transcription factors. Previous crystal structures of BTB domains have revealed tightly interwound homodimers, with the N-terminus from one chain forming a two-stranded anti-parallel beta-sheet with a strand from the other chain. We have solved the crystal structures of the BTB domains from Fanconi anemia zinc finger (FAZF) and Miz1 (Myc-interacting zinc finger 1) to resolutions of 2.0 A and 2.6 A, respectively. Unlike previous examples of BTB domain structures, the FAZF BTB domain is a nonswapped dimer, with each N-terminal beta-strand associated with its own chain. As a result, the dimerization interface in the FAZF BTB domain is about half as large as in the domain-swapped dimers. The Miz1 BTB domain resembles a typical swapped BTB dimer, although it has a shorter N-terminus that is not able to form the interchain sheet. Using cysteine cross-linking, we confirmed that the promyelocytic leukemia zinc finger (PLZF) BTB dimer is strand exchanged in solution, while the FAZF BTB dimer is not. A phylogenic tree of the BTB fold based on both sequence and structural features shows that the common ancestor of the BTB domain in BTB-ZF (bric à brac, tramtrack, broad-complex zinc finger) proteins was a domain-swapped dimer. The differences in the N-termini seen in the FAZF and Miz1 BTB domains appear to be more recent developments in the structural evolution of the domain.

Correlation of structure and function in oligonuclear zinc(II) model phosphatases.

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.

Synthesis of novel steroid-tetrahydroquinoline hybrid molecules and D-homosteroids by intramolecular cyclization reactions.

Steroidal aryliminium salts were prepared from D-seco-pregnene aldehyde 2b, and their BF3.OEt2-catalyzed reactions were studied. The nature of the substituent R1 in the anilines 3-6 essentially influenced the chemoselectivity. Using unsubstituted 3, 4-methoxy- (4) or 4-bromoaniline (5), different tetrahydroquinoline derivatives 7a-13a via intramolecular hetero Diels-Alder reaction were formed. In the case of 4-nitroaniline (6) the N-arylamino-D-homopregnane (14a) were also obtained. We assume, that an intramolecular Prins reaction led to this type of fluoro-D-homosteroid. The main products represent a new class of tetrahydroquinolino-androstenes.