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 and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely 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 nopaline at similar rates, suggesting that both subunits contributed to the substrate 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 instead of B-A) led to reduced activity.
Zanker
H
H
Institut für Biologie II, Universität Freiburg, Germany.
Lurz
G
G
Langridge
U
U
Langridge
P
P
Kreusch
D
D
Schröder
J
J
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
C008666:nopaline; C005394:octopine; D001120:Arginine; D000587:Oxidoreductases Acting on CH-NH Group Donors; C088599:nopaline oxidase; C088600:octopine oxidase