EXPRESSION CLONING OF A GIBBERELLIN 20-OXIDASE, A MULTIFUNCTIONAL ENZYME INVOLVED IN GIBBERELLIN BIOSYNTHESIS

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is re moved by oxidation to give the C-19-GAs, which include the biologicall y active plant hormones. We report the isolation of a cDNA clone encod ing a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA libra ry from developing cotyledons of pumpkin (Cucurbita maxima L.) for exp ression of this enzyme. When mRNA from either the cotyledons or the en dosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA(12) 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by t ryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in lambda gt11 was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was conf irmed by the demonstration of GA(12) 20-oxidase activity in single bac teriophage plaques. Recombinant protein from a selected clone catalyze d the three-step conversions of GA(12) to GA(25) and of GA(53) to GA(1 7), as well as the formation of the C-19-GAs, GA(1), GA(9), and GA(20) , from their respective aldehyde precursors, GA(23), GA(24), and GA(19 ). The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The p redicted M(r) (43,321) and pI (5.3) are similar to those determined ex perimentally for the native GA 20-oxidase. Furthermore, the derived am ino acid sequence includes sequences obtained from the N terminus and two tryptic peptides from the native enzyme. It also contains regions that are highly conserved in a group of non-heme Fe-containing dioxyge nases.