Purification, crystallization and preliminary X-ray diffraction analysis of a fungal saponin-detoxifying enzyme.
Tomatinase, an extracellular enzyme belonging to family 3 of the glycosyl hydrolases, is produced by the fungal tomato-leaf pathogen Septoria lycopersici and detoxifies the saponin alpha-tomatine. An efficient strategy for purification of the enzyme from fungal culture medium has been developed. Single crystals have been grown by vapour diffusion at 289 K from 17.5%(w/v) PEG 4K, 5%(v/v) 2-propanol and 0.1 M sodium acetate pH 4.5 as precipitant. When cryoprotected at 100 K, these crystals diffract to at least 3.0 A and belong to space group P2(1)2(1)2. Based on an estimated molecular weight of 110 kDa for the glycosylated protein and assuming two molecules in the asymmetric unit, the crystals contain approximately 46% solvent.
Bamford
Vicki A
VA
School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, England.
Kolade
Ola O
OO
Osbourn
Anne E
AE
Hemmings
Andrew M
AM
D001203:Ascomycota / Q000201:enzymology*; D003460:Crystallization; D018360:Crystallography, X-Ray; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D018551:Solanum lycopersicum / Q000382:microbiology*; D012503:Saponins / Q000037:antagonists & inhibitors* / Q000633:toxicity
D012503:Saponins; D006026:Glycoside Hydrolases; C099475:tomatinase