PURIFICATION AND CHARACTERIZATION OF BETA(2)-TOMATINASE, AN ENZYME INVOLVED IN THE DEGRADATION OF ALPHA-TOMATINE AND ISOLATION OF THE GENE ENCODING BETA(2)-TOMATINASE FROM SEPTORIA-LYCOPERSICI

Lycopersicon species often contain the toxic glycoalkaloid alpha-tomat ine, which is proposed to protect these plants from general microbial infection, However, fungal pathogens of tomato often are tolerant to a lpha-tomatine and detoxification of alpha-tomatine may be how these pa thogens avoid this potential barrier, As an initial step to evaluate t his possibility, we have purified to homogeneity a beta-1,2-D glucosid ase from the tomato pathogen Septoria lycopersici that hydrolyzes the beta-1,2-D glucosyl bond on the tetrasaccharide moiety of alpha-tomati ne to produce beta(2)-tomatine. The enzyme is a 110-kDa protein with a pI of 4.5 and a K-m for alpha-tomatine of 62 mu M, Little or no activ ity was detected on a variety of other glycosides. The gene encoding t his protein was isolated and contains an open reading frame of 803 ami no acids that shares sequence homology with several other beta-D-gluco sidases. When S. lycopersici was incubated with alpha-tomatine, beta(2 )-tomatinase mRNA accumulated, suggesting that the enzyme is substrate inducible, Aspergillus nidulans expressed ''beta(2)-tomatinase'' acti vity when transformed with this gene but transformants were still sens itive to alpha-tomatine.