Induction and purification of kievitone hydratase from Fusarium solani f. sp. phaseoli.
Fusarium solani f. sp. phaseoli is capable of detoxifying the major isoflavonoid phytoalexins produced by its host plant Phaseolus vulgaris. One of the enzymic activities involved is kievitone hydratase (KHase), a secreted glycoprotein which catalyses the conversion of kievitone to the less fungitoxic derivative, kievitone hydrate. Even under conditions of substrate induction, the enzyme is expressed at levels that are too low for satisfactory purification. Therefore, several other isoflavonoids were tested as inducers in culture. Among the phytoalexins produced by the host plant, phaseollinisoflavan was the best inducer, elevating the level of secreted enzyme eight-fold. Treatment with biochanin A, a product of chickpea, resulted in a 16-fold increase of secreted activity. The maximum rate of induction was observed 9-24 hr after addition of biochanin A, during which time several metabolites of the inducer were also present. KHase was purified from filtrates of biochanin A-induced cultures. Denaturing gel electrophoresis indicated that two species of Mr 47,000 and 49,000 copurified with the activity. N-Terminal sequence analysis indicated that the two species possessed related, or identical, polypeptide moieties. Comparison with the size of the non-denatured enzyme, previously determined to be ca 100,000, indicates that its native state is a dimer.
Turbek
C S
CS
Department of Plant Pathology, University of Kentucky, Lexington 40546-0091.
Li
D X
DX
Choi
G H
GH
Schardl
C L
CL
Smith
D A
DA
D000595:Amino Acid Sequence; D004790:Enzyme Induction; D005419:Flavonoids / Q000378:metabolism / Q000494:pharmacology; D005670:Fusarium / Q000201:enzymology*; D006836:Hydro-Lyases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010936:Plant Extracts / Q000378:metabolism*; D012717:Sesquiterpenes; D013729:Terpenes; D000094162:Phytoalexins
D005419:Flavonoids; D010936:Plant Extracts; D012717:Sesquiterpenes; D013729:Terpenes; D006836:Hydro-Lyases; D000094162:Phytoalexins