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Title: DESOXYHEMIGOSSYPOL O-METHYLTRANSFERASE FROM COTTON STEMS

Author
item LIU, JINGGAO
item BENEDICT, C - TEXAS A&M UNIVERSITY
item Stipanovic, Robert - Bob
item Bell, Alois - Al

Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Proceedings
Publication Acceptance Date: 1/9/1998
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Terpenoid aldehydes and naphthofuran precursors are synthesized in diseased tissues as part of an active defense mechanism. The terpenoid aldehyde hemigossypol (HG) is derived by oxidation of the terpenoid naphthofuran desoxyhemigossypol (dHG). The enzyme dHG O-methytransferase (dHG OMT) catalyses the transfer of a methyl group from S-adenosyl-L-methionine to dHG to form desoxyhemigossypol-6-methyl ether (dMHG). dMHG can undergo th same oxidation as dHG to yield the methylated derivative of HG (i.e., hemigossypol-6-methyl ether). The methylated terpenoids are only about one-half as toxic as the unmethylated parents to microbial pathogens. Antisense constructs of dHG OMT gene may prevent the conversion of dHG to dMHG by blocking the synthesis of the enzyme, and thereby increase resistance of the cotton plant to pathogens. dHG OMT was purified to homogeneity from the cotton stele tissue inoculated with Verticillium dahliae by a Q-Sepharose anion exchanges column, an Ultrogel AcA34 gel filtration column, a 3-hydroxy-4-methoxyphenethylamine-Sepharose affinity column, a 3,4-dimethoxyphenethylamine-Sepharose affinity column, and a 2¿,3¿-ADP-Sepharose 4B affinity column. An overall 851 fold purification has been achieved. The purified enzyme showed a single band at 41.2 kDa. on SDS-PAGE. It had a native molecular weight of 81.4 kDa. and consisted of two identical subunits. The enzyme exhibited high substrate specificity. Substrate-saturation kinetic data were obtained with the dHG OMT preparations purified to homogeneity, and were typical Michaelis-Menten type. A Km of 4.6 uM and a km/kcat of 5.08 x 104 s-1(mol/L)-1 were determined for dHG and a km of 81.4 um and a km/kcat of 1.83 x 103 s-1 (mol/L)-1 were determined for SAM.