is a glycoside hydrolase
Glycoside hydrolases catalyze the hydrolysis of the glycosidic linkage to release smaller sugars...
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
that catalyzes the conversion of trehalose
Trehalose, also known as mycose or tremalose, is a natural alpha-linked disaccharide formed by an α,α-1,1-glucoside bond between two α-glucose units. In 1832, H.A.L. Wiggers discovered trehalose in an ergot of rye, and in 1859 Marcellin Berthelot isolated it from trehala manna, a substance made...
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
. It is found in most animals.
The non-reducing disaccharide trehalose (α-D-glucopyranosyl-1,1-α-D-glucopyranoside) is one of the most important storage carbohydrates, which is present in almost all forms of life except mammals. The disaccharide is hydrolyzed into two molecules of glucose by the enzyme trehalase. There are two
types of trehalases found in Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
, viz. neutral trehalase (NT) and acid trehalase (AT) classified according to their pH optima . NT has an optimum pH of 7.0, while that of AT is 4.5.
Recently it has been reported that more than 90% of total AT activity in S. cerevisiae is extracellular and cleaves extracellular trehalose into glucose in the periplasmic space.
One molecule of trehalose is hydrolyzed to two molecules of glucose by the enzyme trehalase. Enzymatic hydrolysis of trehalose was first observed in Aspergillus niger
Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food...
by Bourquelot in 1893. Fischer reported this reaction in S. cerevisiae
in 1895. Since then the trehalose hydrolyzing enzyme, trehalase (α, α-trehalose-1-C-glucohydrolase, EC 18.104.22.168) has been reported from many other organisms including plants and animals. Though trehalose is not known to be present in mammals, trehalase enzyme is found to be present in the kidney brush border membrane and the intestinal villi membranes. In the intestine the function of this enzyme is to hydrolyze ingested trehalose. Individuals with a defect in their intestinal trehalase have diarrhea when they eat foods with high trehalose content, such as mushrooms . Trehalose hydrolysis by trehalase enzyme is an important physiological process for various organisms, such as fungal spore germination, insect flight, and the resumption of growth in resting cells.
Trehalose has been reported to be present as a storage carbohydrate in Pseudomonas
Pseudomonas is a genus of gammaproteobacteria, belonging to the family Pseudomonadaceae containing 191 validly described species.Recently, 16S rRNA sequence analysis has redefined the taxonomy of many bacterial species. As a result, the genus Pseudomonas includes strains formerly classified in the...
Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the division Firmicutes. Bacillus species can be obligate aerobes or facultative anaerobes, and test positive for the enzyme catalase. Ubiquitous in nature, Bacillus includes both free-living and pathogenic species...
Rhizobium is a genus of Gram-negative soil bacteria that fix nitrogen. Rhizobium forms an endosymbiotic nitrogen fixing association with roots of legumes and Parasponia....
and in several actinomycetes and may be partially responsible for their resistance properties. Most of the trehalase enzymes isolated from bacteria have as optimum pH of 6.5-7.5. The trehalase enzyme of Mycobacterium smegmatis is a membrane bound protein . Periplasmic trehalase of Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
K12 is induced by growth at high osmolarity . The hydrolysis of trehalose into glucose takes place in the periplasm, and the glucose is then transported into the bacterial cell. Another cytoplasmic trehalase has also been reported from E. coli
. The gene, which encodes this cytoplasmic trehalase, exhibits high homology to the periplasmic trehalase.
Trehalase in plants
In plant kingdom, though trehalose has been reported from several pteridophytes including Selaginella lepidophylla
Selaginella lepidophylla is a species of desert plant in the spikemoss family . S. lepidophylla is noted for its ability to survive almost complete desiccation; during dry weather in its native habitat, its stems curl into a tight ball and uncurl when exposed to moisture...
and Botrychium lunaria
; the sugar is rare in vascular plants and reported only in ripening fruits of several members of Apiaceae
The Apiaceae , commonly known as carrot or parsley family, is a group of mostly aromatic plants with hollow stems. The family is large, with more than 3,700 species spread across 434 genera, it is the sixteenth largest family of flowering plants...
and in the leaves of the desiccation-tolerant angiosperm Myrothamnus flabellifolius
. But, the enzyme trehalase is ubiquitous in plants. This is puzzling that trehalase is present in higher plants, though its substrate is absent. No clear role has been demonstrated for trehalase activity in plants. It has been suggested that trehalases could play a role in defense mechanisms or the enzyme could play a role in the degradation of trehalose derived from plant-associated microorganisms.
In S. cerevisiae
at least two distinct trehalases have been reported. One was reported to be regulated by cAMP
Cyclic adenosine monophosphate is a second messenger important in many biological processes...
Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation activates or deactivates many protein enzymes....
, . This enzyme activity was found in the cytosol. A second trehalase activity was found in the vacuoles of the same oraganism12. The pH optimum of cytosolic trehalse was found to be about 7.0 and thus, it was referred as neutral trehalase (NT); while the vacuolar trehalase enzyme was reported to be most active at pH around 4.5 and was termed as acid trehalase (AT). These two enzymes are encoded by two different genes – NTH1 and ATH1 respectively.
The cytosoilc trehalase enzyme, NT, has been purified and characterized extensively from S. cerevisiae
. In non-denaturing gels this enzyme protein exhibited a molecular mass of 160 kDa, while in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
) it showed a mass of 80 kDa4. This hydrolase enzyme is specific for trehalose. The Km of NT has been reported to be 5.7mM. The gene responsible for trehalase activity in S. cerevisiae
is NTH1. This gene is with an open reading frame of 2079 base pairs (bp), encoding a protein of 693 amino acids, corresponding to a molecular mass of 79569 Da.
NT activity is regulated by protein phosphorylation-dephosphorylation. Phosphorylation with cAMP-dependent protein kinase activates NT. Dephosphorylation of the purified phosphorylated enzyme by alkaline phosphatase caused an almost complete inactivation of the enzyme activity; but a recovery of the enzyme activity could be observed by rephosphorylation while incubating with ATP and protein kinase. The activity of NT in crude extracts is enhanced by polycations, while the activity of purified phosphorylated NT is inhibited by them. The activation of crude extracts was found to be due to the removal of polyphosphates, both of which inhibit NT activity.
The molecular weight of AT was found to be 218 kDa by gel filtration chromatography. AT is a glycoprotein
Glycoproteins are proteins that contain oligosaccharide chains covalently attached to polypeptide side-chains. The carbohydrate is attached to the protein in a cotranslational or posttranslational modification. This process is known as glycosylation. In proteins that have segments extending...
. It has 86% carbohydrate content. It has been reported that the maturation of AT is a stepwise process beginning with a carbohydrate-free 41 kDa protein; this form is then core-glycosylated in the endoplasmic reticulum to form a 76 kDa glycol-protein. In Golgi bodies, the protein is further glycosylated yielding a 180 kDa form, which ultimately attains a maturity in vacuoles, where its molecular weight becomes around 220 kDa . The 41 kDa carbohydrate free protein moiety of the enzyme was obtained by Endoglycosidase H
The enzyme Endoglycosidase H The enzyme Endoglycosidase H The enzyme Endoglycosidase H (Endo-β-N-acetylglucosaminidase H, is a highly specific endoglycosidase which cleaves asparagine-linked mannose rich oligosaccharides, but not highly processed complex oligosaccharides from glycoproteins...
treatment of purified AT, resulted after sodium dodecyl sulfate gel electrophoresis27. AT exhibited an apparent Km for trehalose of about 4.7 mM at pH 4.5. The gene responsible for AT activity in S. cerevisiae
Ath1p, i.e. AT, has been reported to be necessary for S. cerevisiae
to utilize extracellular trehalose as carbon source16. ATH1 deletion mutant of the yeast could not grow in the medium with trehalose as the carbon source.
Researchers have suggested that AT moves from its site of synthesis to the periplasmic space, where it binds exogenous trehalose to internalize it and hydrolyze it in the vacuoles . Recently it has been shown that more than 90% of AT activity in S. cerevisiae
is extracellular and the hydrolysis of trehalose into glucose takes place at the periplasmic space. Previously, a highly glycosylated protein, gp37, which is the product of YGP1 gene, was reported to be co-purified with AT activity . Invertase activity was also reported to be co-purified with AT activity . The physical association of AT with these two proteins was thought to suffice for the AT to be secreted by invertase and gp37 secretion pathways in absence of any known secretion signal for Ath1p.
In a Candida utils
strain, one regulatory a one non-regulatory trehalase were also reported . These two enzymes were reported to be distinguishable by their molecular weight, behavior in ion-exchange chromatography and kinetic properties. The regulatory trehalase appeared to be a cytoplasmic enzyme and the nonregulatory enzyme was mostly detected in vacuoles. But, in a more recent report, a C. utils
strain was demonstrated to lack any detectable AT activity but contain only NT activity . AT activity was not detectable in this strain, though the strain was shown to utilize extracellular trehalose as carbon source.