Hydroxylation of ent-kaurenoic acid to steviol in Stevia rebaudiana Bertoni--purification and partial characterization of the enzyme.
The diterpenoic compound steviol (ent-kaur-16-en-13-ol-19-oic acid) is the aglycone of sweet glycosides accumulated in Stevia rebaudiana Bertoni. This compound is the hydroxylated form of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA). The hydroxylation of ent-KA to form steviol requiring NADPH and molecular oxygen was detected in stroma prepared from S. rebaudiana Bertoni. The enzyme was purified from leaf extract to apparent homogeneity with a molecular mass of 39 kDa. Taken together with the value of 160 kDa estimated for native enzyme, this suggested that the hydroxylating enzyme is a homotetramer. The N-terminal sequence was determined through 20 residues. The pH optimum was 7.5-7.8. Apparent Km values were 11.1 microM for ent-KA and 20.6 microM for NADPH. Its visible absorption spectrum suggested that the enzyme was flavoprotein. The stoichiometric relationship between the formation of steviol and the utilization of ent-KA and cofactors confirmed the equation ent-KA + NADPH + H(+) + O2-->steviol + NADPH(+) + H2O.
Kim
K K
KK
Department of Life Science and Biotechnology, Faculty of Life and Environmental Science, Shimane University, Japan.
Sawa
Y
Y
Shibata
H
H
D000595:Amino Acid Sequence; D004224:Diterpenes / Q000378:metabolism*; D045786:Diterpenes, Kaurane; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D010944:Plants / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D013549:Sweetening Agents / Q000378:metabolism
D004224:Diterpenes; D045786:Diterpenes, Kaurane; D013549:Sweetening Agents; C012042:steviol; D009249:NADP; C051436:kaurenoic acid; D006899:Mixed Function Oxygenases; C101466:ent-kaurenoic acid 13-hydroxylase; D010100:Oxygen