Molecular characterisation of gibberellin 20‐oxidases. Structure‐function studies on recombinant enzymes and chimaeric proteins

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Abstract
Gibberellin (GA) 20‐oxidases are multifunctional enzymes that catalyse reactions at an important branch point in the GA biosynthetic pathway. These enzymes oxidise the C‐20 methyl group of a diterpene carboxylic acid precursor (e.g. GA12) to form an alcohol (in our case GA15‐open lactone) and an aldehyde (GA24). The aldehyde is either oxidised to a tricarboxylic acid (GA25) or, with loss of carbon‐20 and lactonisation, to a C19‐GA (GA9). This branching is interesting to study, because C19‐GA derivatives function as plant hormones in different tissues, whereas the C20‐GA tricarboxylic acids have no known function. We have constructed chimaeric proteins by combining a GA 20‐oxidase from immature seeds of Cucurbita maxima L., which produces mainly C‐20 carboxylic acids, with a 20‐oxidase from Marah macrocarpus immature seeds, which forms predominantly CC19‐GAs. The cDNAs encoding these two very similar 20‐oxidases were digested with restriction endonucleases Van 911. Bcl 1, and Bsa WI, and six chimaeric sequences were produced by recombination of the DNA fragments. The pCM1 ‐construct was obtained by exchanging nt 303–809 of the Cucurbita cDNA with the homologous DNA from the March 20‐oxidase. In pCM2, pCM3, pCM4, pCM5 and pCM6, nt 810–992, nt 993–end, nt 303–992, nt 810–end, and nt 311–end were exchanged, respectively. All constructs were cloned in a pUC18 vector and functionally expressed in E. coli NM522 cells. GA 20‐oxidase activity was detectable in cell‐lysates from the transformed E. coli, but the extent and kind of conversion depended on the construct. Highest conversion of GA12was found with pCM1 and pCM3, one‐tenth of this conversion was observed with pCM5 and pCM6, and one‐hundredth was obtained with the hybrid proteins from pCM2 and pCM4. With pCM2 and pCM4, neither the C19‐end product, GA9, nor the C20‐end product, GA25‐was formed. However, after transformation with constructs pCM1, pCM3, pCM5 or pCM6. GA9accounted for 30, 40, 60 and 90%, respectively, of the end products formed. Thus, the segments originating from M. macrocarpus conferred upon the chimaeric proteins an increasing ability to direct the biosynthetic flow into C19‐GAs in this order. Although GA24is the immediate precursor, much less end products were formed by using this substrate.