Mikimopine synthase (mis) gene on pRi1724

By determination of the nucleotide sequence adjacent to the right border of T-DNA of the mikimopine-type Ri plasmid (pRi1724) in Agrobacterium rhizoge nes, a new open reading frame (ORF) encoding 318 amino acids was found. A t ranscript of 1.35 kb derived from this ORF was observed in hairy roots of A juga reptans by northern blotting analysis. Including its own promoter and terminator, this ORF was isolated from the pRi1724 T-DNA and introduced int o tobacco plants by the Agrobacterium-binary vector system. Since mikimopin e, an opine and a stereoisomer of cucumopine, was accumulated in all organs of the transgenic tobacco plants, the new ORF was deduced to be the mikimo pine synthase gene. For comparison, the nucleotide sequence of cucumopine s ynthase encoded on pRi2659 was also determined. No homology was found betwe en mikimopine synthase and cucumopine synthase at the nucleotide, but parti al homology was found at the amino acid level. Mikimopine synthase and cucu mopine synthase produced by a protein expression system using E. coli catal yzed the synthesis of mikimopine and cucumopine from L-histidine and a-keto glutaric acid, requiring NADH as a cofactor. These synthesized opines were identified by paper electrophoresis, TLC and HPLC analyses. The synthesized mikimopine or cucumopine could be degraded by A. rhizogenes strains harbor ing Ri plasmids encoding the respective catabolic enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.