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Title: BIOSYNTHESIS OF GOSSYPOL: PURIFICATION AND PROPERTIES OF DESOXYHEMIGOSSYPOL 6-O-METHYLTRANSFERASE

Author
item LIU, J - TEXAS A&M UNIVERSITY
item BENEDICT, C - TEXAS A&M UNIVERSITY
item ALCHANATI, I - TEXAS A&M UNIVERSITY
item Stipanovic, Robert - Bob

Submitted to: National Cotton Council Beltwide Cotton Conference
Publication Type: Abstract Only
Publication Acceptance Date: 1/9/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Desoxyhemigossypol 6-O-methyltransferase (dHG 6-OMT) was partially purified from the cotton stele tissue inoculated with Verticillium dahliae by ultracentrifugation, an ion exchange chromatography, and gel filtration chromatography on a FPLC column. The enzyme which was purified 267-fold catalyzed the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the 6-hydroxy group of desoxyhemigossypol (dHG), one of the several sesquiterpenoid phytoalexins induced in response to the fungal attack. The enzymatic methylation of dHG was established with radioactive [methyl-14C]-SAM. The biosynthetic products were separated by HPLC and desoxyhemigossypol 6-methyl ether (dMHG) was identified as the methylated product. This finding was directly confirmed by a feeding experiment with [methyl-2H3]-SAM using a purified enzyme preparation. The enzymatically labeled deuterated phytoalexins were separated by HPLC. The fraction corresponding to dMHG had an identical UV spectrum with the authentic dMHG. This fraction was analyzed by GC-MS and the major GC peak was identified as deuterated dMHG. The purified dHG 6-OMT had a native molecular mass of 81 kD. The enzyme did not require a divalent cation for activity and several divalent cations such as Cu2+, CO2+, Zn2+, and Mn2+ appreciably inhibited the enzyme activity. Thiol blocking reagents also strongly inhibited the enzyme activity, indicating possible involvement of thiol groups in the active center of the enzyme. Substrate-saturation kinetics of the purified enzyme for dHG and SAM were typical Michaelis-Menten type with Km values of 4.5 and 20.8 uM, respectively.