Expression cloning of a gibberellin 20-oxidase, a multifunctional enzyme involved in gibberellin biosynthesis.

Abstract

In the biosynthetic pathway to the gibberellins (GAs), carbon-20 is removed by oxidation to give the C19-GAs, which include the biologically active plant hormones. We report the isolation of a cDNA clone encoding a GA 20-oxidase [gibberellin, 2-oxoglutarate:oxygen oxidoreductase (20-hydroxylating, oxidizing) EC 1.14.11.-] by screening a cDNA library from developing cotyledons of pumpkin (Cucurbita maxima L.) for expression of this enzyme. When mRNA from either the cotyledons or the endosperm was translated in vitro using rabbit reticulocyte lysates, the products contained GA12 20-oxidase activity. A polyclonal antiserum was raised against the amino acid sequence of a peptide released by tryptic digestion of purified GA 20-oxidase from the endosperm. A cDNA expression library in Agtll was prepared from cotyledon mRNA and screened with the antiserum. The identity of positive clones was confirmed by the demonstration of GA12 20-oxidase activity in single bacteriophage plaques. Recombinant protein from a selected clone catalyzed the three-step conversions of GA12 to GA2s and of GA53 to GA17, as well as the formation of the C,,-GAs, GA1, GA,, and GA20, from their respective aldehyde precursors, GA23, GA2,4, and GA19. The nucleotide sequence of the cDNA insert contains an open reading frame of 1158 nt encoding a protein of 386 amino acid residues. The predicted Mr (43,321) and pI (5.3) are similar to those determined experimentally for the native GA 20-oxidase. Furthermore, the derived amino 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 dioxygenases.The gibberellins (GAs) form a large group of diterpenoid natural products. Certain GAs function as hormones in plants, controlling many aspects of development, including stem extension, fruit set, and seed germination (1). The later steps of the GA biosynthetic pathway are catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which oxidize the GA skeleton at the 20, 3,8, and 2p3 positions (see Fig.

Cloning gibberellin dioxygenase genes from pumpkin endosperm by heterologous expression of enzyme activities in Escherichia coli

“…The catalytic properties of recombinant 20-oxidase were exactly the same as previously found for native 20-oxidase from pumpkin endosperm (6) and recombinant 20-oxidase from pumpkin cotyledons (7). These three enzymes catalyze the successive oxidation of the C-20 methyl group of [ 14 53 and produce C 19 GAs in low yields with both precursors (data not shown).…”

“…26). There are many reports on the expression of recombinant enzymes retaining catalytic activity as fusion proteins (7)(8)(9)(10)(11)(12)(13)(14)(15)(26)(27)(28)(29). Therefore, the method described here should be applicable for cloning a wide range of enzymes, provided that these are not involved in the metabolism of or are toxic to E. coli itself.…”

“…To identify the most advantageous starting material, poly(A) ϩ RNA was extracted from endosperm of immature seeds without developed cotyledons, with cotyledons of 20%, and with cotyledons of 40% the length of the seed lumen (17) and translated in vitro using rabbit reticulocyte lysate (7). The products were directly assayed for GA dioxygenase activities by incubation with [ 14 25 (250 Bq each, 40 pmol) and extraction as described (7). These precursors would assure detection of GA 7-oxidase, 20-oxidase, and 3␤-hydroxylase, respectively.…”

“…The coding region of clone E5 is different at seven positions (176, 502, 545, 672, 674, 931, and 1098) from the 20-oxidase previously cloned from pumpkin cotyledons (ref. 7, not illustrated in Fig. 3), resulting in changes of the amino acids 174 (Pro to Ser), 216 (Phe to Tyr), 217 (Glu to Lys), and 358 (Asp to Ala).…”

“…Recently, the structural gene for a GA 20-oxidase was cloned from pumpkin seed cotyledons by immunoscreening (7). The cloning of another 20-oxidase gene from pumpkin endosperm was reported, but details of this work were not documented (8).…”