Isolation and characterization of chitin from bumblebee (Bombus terrestris).

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.

Pectin methylesterases: sequence-structural features and phylogenetic relationships.

Pectin methylesterases (PMEs) are enzymes produced by bacteria, fungi and higher plants. They belong to the carbohydrate esterase family CE-8. This study deals with comparison of 127 amino acid sequences of this family containing the five characteristic sequence segments: 44_GxYxE, 113_QAVAL, 135_QDTL, 157_DFIFG, 223_LGRPW (Daucus carota numbering). Six strictly conserved residues (Gly44, Gly154, Asp157, Gly161, Arg225 and Trp227) and six conservative ones (Ile39, Ser86, Ser137, Ile152, Ile159 and Leu223) were identified. A set of 70 representative PMEs was created. The sequences were aligned and the evolutionary tree based on the alignment was calculated. The tree reflected the taxonomy: the fungal and bacterial PMEs formed their own clusters and the plant enzymes were grouped into eight separate clades. The plant PME from Vitis riparia was placed in a common clade with fungi. Three plant clades (Plant 1, 2 and 3) were relatively homogenous reflecting high degree of mutual sequence identity. The clade Plant 4 contained PMEs from flower parts (mostly form pollen) and was heterogenous, like the clades Plant 1a and 2a, which moreover exhibit an intermediate character. The clades Plant X1 and X2 were situated in the tree close to microbial clades and represented atypical plant PMEs. Taking into account the remaining plant PMEs, an expanded plant alignment and tree (with most Arabidopsis thaliana and Oryza sativa enzymes), were prepared. An exclusive Arabidopsis alignment and tree indicated the existence of a new plant clade X3. In the pre pro region of most plant enzymes a longer conserved segment containing basic dipeptide, R(K)/R(K), that precedes the N-terminal end of PME was revealed. This was not observed in the clade Plant X1 and majority of the clade Plant X2. This study brings further the description of occurrence of potential glycosylation sites in pre pro sequences and in mature enzymes as well as important amino acid residues, such as aspartates, cysteines, histidines and other aromatic residues (Tyr, Phe and Trp), with discussion of their possible function in the activity of PMEs.

Crystal structure of plant pectin methylesterase.

Pectin is a principal component in the primary cell wall of plants. During cell development, pectin is modified by pectin methylesterases to give different properties to the cell wall. This report describes the first crystal structure of a plant pectin methylesterase. The beta-helical structure embodies a central cleft, lined by several aromatic residues, that has been deduced to be suitable for pectin binding. The active site is found at the center of this cleft where Asp157 is suggested to act as the nucleophile, Asp136 as an acid/base and Gln113/Gln135 to form an anion hole to stabilize the transition state.