OCTOPINE AND NOPALINE OXIDASES FROM TI PLASMIDS OF AGROBACTERIUM-TUMEFACIENS - MOLECULAR ANALYSIS, RELATIONSHIP, AND FUNCTIONAL-CHARACTERIZATION

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine an d nopaline in Agrobacterium spp. The first enzymatic step is the oxida tive cleavage into L-arginine and pyruvate or 2-ketoglutarate, respect ively, by membrane-bound opine oxidases requiring two polypeptides (su bunits B and A) for function. The DNA sequences showed that the subuni ts of pTiAch5 and pTiC58 are related, but none of the proteins reveale d significant similarities to the biosynthetic enzymes expressed in tr ansformed plant cells. The four proteins had no extensive overall simi larity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, fl avin mononucleotide, or NAD(P)(+) as cofactors. However, the activitie s were completely independent of added cofactors, and the nature of th e electron acceptor remained unclear. Membrane solubilization led to c omplete loss of enzyme activity. The nopaline oxidase accepted nopalin e and octopine (V-max ratio, 5:1) with similar K-m values (1.1 mM). Th e octopine oxidase had high activity with octopine (K-m = 1 mM) and ba rely detectable activity with nopaline, The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB -ooxA both produced active enzymes which oxidized octopine and nopalin e at similar rates, suggesting that both subunits contributed to the s ubstrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B inst ead of B-A) led to reduced activity.