Insights into the structure and function of fungal ß-mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme.

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.

[Mannanases of microorganisms].

The current data on microbial mannanases are summarised in the work. The questions concerning the spreading of mannanases substrates--mannans in nature, their structural variations are regarded. The production of mannanases by micro-organisms of different taxonomic groups, their physico-chemical properties and substrate specificity are described. The ways of mannans hydrolysis by mannanase are estimated. The questions of cloning and expression of mannanase genes have been discussed. These results are very important for producing highly effective microbial biosynthetics.

Expression and location of endo-beta-mannanase during the ripening of tomato fruit, and the relationship between its activity and softening.

Endo-beta-mannanase is thought to play a role in tomato fruit ripening by participating in the degradation of cell walls. Its spatial and temporal expression during ripening was examined, as was the relationship between its activity and softening of the fruit using a large number of tomato lines, and by suppression of transcription of the endo-beta-mannanase (LeMan4a) gene. Immunolocalization studies showed that the enzyme is expressed in the fruit cell wall at all ripening stages, but it is not active during the initial green stage; this is not due to the presence of inhibitors of its activity, nor due to changes in its mRNA sequence. Transient expression in onion epidermal cells of endo-beta-mannanase transcripts fused to green fluorescent protein resulted in the expressed enzyme being localized to the cell walls. Transgenic tomato plants expressing a GUS gene attached to the LeMan4a promoter showed that this occurs initially during ripening in the skin and outer pericarp of the fruit, and later in the skin and throughout the pericarp. Fruit firmness and activity of endo-beta-mannanase were not strongly correlated during ripening of many lines of tomato. Several plants of cv. Micro-Tom were transformed using RNA interference (mRNAi) and antisense RNA strategies to suppress transcription of the LeMan4a gene. When endo-beta-mannanase activity was much reduced in the transgenic fruits, their firmness was higher compared to those of control fruits at the turning and orange-color stages, but at the red-ripe stage firmness was similar between the two fruit types. We suggest that while the enzyme does participate in fruit ripening it alone is not sufficient to cause hydrolysis of the cell walls which results in their weakening; it likely plays a cooperative role with other known wall-modifying enzymes, and/or is involved in cell wall rearrangement.

Germination ecophysiology of Annona crassiflora seeds.

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.

The emergence of embryos from hard seeds is related to the structure of the cell walls of the micropylar endosperm, and not to endo-beta-mannanase activity.

BACKGROUND AND AIMS: Seeds of carob, Chinese senna, date and fenugreek are hard due to thickened endosperm cell walls containing mannan polymers. How the radicle is able penetrate these thickened walls to complete seed germination is not clearly understood. The objective of this study was to determine if radicle emergence is related to the production of endo-beta-mannanase to weaken the mannan-rich cell walls of the surrounding endosperm region, and/or if the endosperm structure itself is such that it is weaker in the region through which the radicle must penetrate. METHODS: Activity of endo-beta-mannanase in the endosperm and embryo was measured using a gel assay during and following germination, and the structure of the endosperm in juxtaposition to the radicle, and surrounding the cotyledons was determined using fixation, sectioning and light microscopy. KEY RESULTS: The activity of endo-beta-mannanase, the major enzyme responsible for galactomannan cell wall weakening increased in activity only after emergence of the radicle from the seed. Thickened cell walls were present in the lateral endosperm in the hard-seeded species studied, but there was little to no thickening in the micropylar endosperm except in date seeds. In this species, a ring of thin cells was visible in the micropylar endosperm and surrounding an operculum which was pushed open by the expanding radicle to complete germination. CONCLUSIONS: The micropylar endosperm presents a lower physical constraint to the completion of germination than the lateral endosperm, and hence its structure is predisposed to permit radicle protrusion.

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A.

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.