Properties of β-thioglucoside hydrolases (TGG1 and TGG2) from leaves of Arabidopsis thaliana.
Myrosinases (β-thioglucoside glucohydrolase, TGG; EC 3.2.1.147) catalyze the hydrolysis of glucosinolates, a structurally distinct group of nitrogen- and sulfur-containing secondary metabolites, to give a chemically unstable intermediate, glucose and sulfate. This catalysis initiates a chemical defense in crucifer plants as a response to the tissue-damaging activities of herbivores and pathogens. To characterize the individual and collective biochemical properties of the myrosinase enzymes found in the aerial tissues of Arabidopsis thaliana, we purified TGG1 and TGG2, which share 73% amino acid identity, individually from T-DNA insertion lines of Arabidopsis using lectin affinity and anion exchange chromatography. Electrophoresis under denaturing conditions and the mobility of nondenatured TGG1 and TGG2 protein on gel filtration chromatography indicated that the native proteins exist as dimers of 150 and 126 kDa, respectively. Despite their relatively similar kinetic parameters, both enzymes had distinct physicochemical properties such as extractability in low ionic strength buffer and electrophoretic mobility following deglycosylation treatment. Deglycosylation under nondenaturing conditions had limited effects on TGG1 and no effect on TGG2 activity. Both enzymes functioned across a broad range of temperatures (up to 60 °C) and pH values (5-10). These results demonstrate that myrosinases have the ability to function in environments like the digestive tract of insect herbivores that are significantly different from the environment in a damaged plant.
Department of Biological Sciences, Virginia Tech, Blacksburg, VA 24061, USA.
Changhe
C
Zhou
James G
JG
Tokuhisa
David R
DR
Bevan
Asim
A
Esen
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001205:Ascorbic Acid / Q000494:pharmacology; D055162:Biocatalysis / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000201:enzymology*; D011489:Protein Denaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
D029681:Arabidopsis Proteins; D010455:Peptides; D006026:Glycoside Hydrolases; C511862:TGG1 protein, Arabidopsis; C511863:TGG2 protein, Arabidopsis; C022739:thioglucosidase; D001205:Ascorbic Acid