RESUMO
UNLABELLED: Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. ß-Mannosidases are enzymes capable of cleaving nonreducing residues of ß-d-mannose from ß-d-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. ß-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 ß-mannosidase and to refine it to 1.9-Å resolution. Structural comparisons of the T. harzianum GH2 ß-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of ß-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. DATABASE: Coordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum ß-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918.
Assuntos
Proteínas Fúngicas/química , Trichoderma/enzimologia , beta-Manosidase/química , Proteínas de Bactérias/química , Domínio Catalítico , Cristalografia por Raios X , Proteínas Fúngicas/fisiologia , Galactose/análogos & derivados , Glicosilação , Mananas/química , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Homologia Estrutural de Proteína , beta-Manosidase/fisiologiaRESUMO
BACKGROUND AND AIMS: Little is known about environmental factors that break morphophysiological dormancy in seeds of the Annonaceae and the mechanisms involved. The aim of this study was to characterize the morphological and physiological components of dormancy of Annona crassiflora, a tree species native to the Cerrado of Brazil, in an ecophysiological context. METHODS: Morphological and biochemical characteristics of both embryo and endosperm were monitored during dormancy break and germination at field conditions. Seeds were buried in the field and exhumed monthly for 2 years. Germination, embryo length and endosperm digestion, with endo-beta-mannanase activity as a marker, were measured in exhumed seeds, and scanning electron microscopy was used to detect cell division. The effect of constant low and high temperatures and exogenous gibberellins on dormancy break and germination was also tested under laboratory conditions. KEY RESULTS: After burial in April, A. crassiflora seeds lost their physiological dormancy in the winter months with lowest monthly average minimum temperatures (May-August) prior to the first rainfall of the wet season. The loss of physiological dormancy enabled initiation of embryo growth within the seed during the first 2 months of the rainy season (September-October), resulting in a germination peak in November. Embryo growth occurred mainly through cell expansion but some dividing cells were also observed. Endosperm digestion started at the micropylar side around the embryo and diffused to the rest of the endosperm. Exogenous gibberellins induced both embryo growth and endo-beta-mannanase activity in dormant seeds. CONCLUSIONS: The physiological dormancy component is broken by low temperature and/or temperature fluctuations preceding the rainy season. Subsequent embryo growth and digestion of the endosperm are both likely to be controlled by gibberellins synthesized during the breaking of physiological dormancy. Radicle protrusion thus occurred at the beginning of the rainy season, thereby maximizing the opportunity for seedlings to emerge and establish.