Hydroxylation ofent-Kaurenoic Acid to Steviol inStevia rebaudianaBertoni—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 inStevia rebaudianaBertoni. This compound is the hydroxylated form ofent-kaurenoic acid (ent-kaur-16-en-19-oic acid;ent-KA). The hydroxylation ofent-KA to form steviol requiring NADPH and molecular oxygen was detected in stroma prepared fromS. rebaudianaBertoni. 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. ApparentKmvalues were 11.1 μMforent-KA and 20.6 μMfor NADPH. Its visible absorption spectrum suggested that the enzyme was flavoprotein. The stoichiometric relationship between the formation of steviol and the utilization ofent-KA and cofactors confirmed the equation[formula]