A TI PLASMID-ENCODED ENZYME REQUIRED FOR DEGRADATION OF MANNOPINE IS FUNCTIONALLY HOMOLOGOUS TO THE T-REGION-ENCODED ENZYME REQUIRED FOR SYNTHESIS OF THIS OPINE IN CROWN GALL TUMORS

The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid p Ti15955 is related at the nucleotide sequence level to mas1' encoded b y the T region of this plasmid. While Mas1 is required for the synthes is of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi159 55, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb crypti c plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion muta tion in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both str ains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol d ehydrogenase family of oxidoreductases. Lysates prepared from Escheric hia coli cells expressing mocC contained an enzymatic activity that ox idizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the prese nce of NAD(+). The reaction catalyzed by the MOP oxidoreductase is rev ersible; in the presence of NADH, the enzyme reduced SOP to MOP. The a pparent K-m values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl )-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as subs trates for MOP oxidoreductase. These results indicate that mocC encode s an oxidoreductase that, as an oxidase, is essential for the cataboli sm of MOP. The reductase activity of this enzyme is precisely the reac tion ascribed to its T-region-encoded homolog, Mas1, which is responsi ble for biosynthesis of mannopine in crown gall tumors.