Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae.
Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding proteins and myrosinase-associated proteins, is functional in its free form. Characterization of the recombinant MYR1 with respect to pH optimum, substrate specificity, activation by ascorbic acid, and inhibitors showed similar characteristics as previously observed for other plant myrosinases. The indolizidine alkaloid castanospermine, an inhibitor of O-glycosidases, inhibited the hydrolysis of p-hydroxybenzylglucosinolate with a K(i) value of 0.3 microM and 2-deoxy-2-fluoroglucotropaeolin, a specific inhibitor of thioglucosidases, inhibited the enzyme with a K(i) value of 1 mM. The expression of the myrosinase in yeast was transient and the growth of the yeast cells was significantly reduced during the period of expression of the myrosinase. Immunoblot analysis showed that the highest level of expression of MYR1 was obtained 24 h after induction with galactose. The amount of myrosinase protein correlated with the level of enzyme activity. The transient expression of myrosinase indicates that myrosinase is toxic to the cells. This is the first report on successful heterologous expression of a myrosinase and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.
Chen
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Department of Plant Biology, Royal Veterinary and Agricultural University, 40 Thorvaldsensvej, Frederiksberg C, DK-1871, Denmark.
Halkier
B A
BA
D001937:Brassica / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D005822:Genetic Vectors; D005961:Glucosinolates / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D006868:Hydrolysis; D015151:Immunoblotting; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
C097342:4-hydroxybenzylglucosinolate; D005961:Glucosinolates; D010940:Plant Proteins; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C022739:thioglucosidase