CLONING OF ORNITHINE DELTA-AMINOTRANSFERASE CDNA FROM VIGNA-ACONITIFOLIA BY TRANSCOMPLEMENTATION IN ESCHERICHIA-COLI AND REGULATION OF PROLINE BIOSYNTHESIS

Proline prototrophy was restored to an Escherichia coli proBA proline auxotroph by ornithine and a moth-bean (Vigna aconitifolia) cDNA expre ssion library. This novel strategy, ''trans-complementation,'' allowed isolation of a cDNA encoding ornithine delta-aminotransferase (delta- OAT). This enzyme transaminates ornithine to glutamic-gamma-semialdehy de (GSA), thereby bypassing the block in GSA synthesis from glutamate in the proBA mutant. The identity of the mothbean enzyme was confirmed by its high sequence homology to mammalian and yeast delta-OATs as we ll as to a family of bacterial and fungal omega-aminotransferases and an absence of significant homology to various alpha-aminotransferases. The V. aconitifolia OAT cDNA encodes a polypeptide of 48.1 kDa. The n ative enzyme expressed in E. coli appears to be a monomer with K(m) of 2 mM for ornithine and 0.75 mm for alpha-ketoglutarate. Levels of mRN A in V. aconitifolia for DELTA1-pyrroline-5-carboxylate synthetase (P5 CS) and delta-OAT, the two key enzymes for proline synthesis, were mon itored under different physiological conditions. Salt stress and nitro gen starvation induced P5CS mRNA levels and depressed OAT mRNA levels. Conversely, OAT mRNA level was elevated in plants supplied with exces s nitrogen while the P5CS mRNA level was reduced. These data suggest t hat the glutamate pathway is the primary route for proline synthesis i n plants during conditions of osmotic stress and nitrogen limitation w hereas the ornithine pathway assumes prominence under high nitrogen in put.