|Stipanovic, Robert - Bob|
Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/20/2001
Publication Date: N/A
Citation: Interpretive Summary: Gossypol is a compound that occurs naturally in cottonseed and is toxic to some animals including man. As such, it reduces the value of cottonseed. However, gossypol and related compounds are required to protect the plant from diseases and insects. Thus, completely removing gossypol from the cotton plant would be counter productive. We are developing technology to selectively remove gossypol from the cottonseed. Herein, we have studied what compounds the cotton plant uses to produce gossypol and how effectively they are used. One of these compounds, farnesyl diphosphate, is used very efficiently and provides only one product, cadinene. A second compound, nerolidyl diphosphate, is used just as efficiently, but produces several other compounds as well as cadinene. These findings advance our understanding of how cotton and other plants use different intermediate compounds to produce a vast array of chemical compounds including gossypol.
Technical Abstract: The first step in the conversion of the isoprenoid intermediate, farnesyl diphosphate, to sesquiterpene phytoalexins in cotton plants is catalyzed by delta-cadinene synthase. delta-Cadinene is the precursor of desoxyhemigossypol and hemigossypol defense sesquiterpenes. In this paper we have studied the mechanism for the cyclization of farnesyl diphosphate and the putative intermediate, nerolidyl diphosphate, to delta-cadinene. A purified recombinant delta-cadinene synthase cyclizes (1RS)-[1-2H]-(E, E)-farnesyl diphosphate to >98% [5-2H]- and [11-2H]-delta-cadinene. Enzyme reaction mixtures cyclize (3RS)-[4,4,13,13,13-2H5]-nerolidyl diphosphate to 62.1% [8,8,15,15,15-2H5]-delta-cadinene, 15.8% [6,6,15,15,15-2H5] alpha-bisabolol, 8.1% [6,6,15,15,15-2H5]-E-beta- bisabolene, 9.8% [4,4,13,13-2H4]-E-(beta)-farnesene and 4.2% unknowns. Competitive studies show that (3R)-nerolidyl diphosphate is the active enantiomer of (3RS)-nerolidyl diphosphate that cyclized to delta-cadinene. The Kcat/Km values demonstrate that the synthase uses E,E-farnesyl diphosphate as effectively as (3R)-nerolidyl diphosphate in the formation of delta-cadinene. Cyclization studies with (3R)-nerolidyl diphosphate show that the formation of delta- cadinene, E-(beta)-farnesene and beta-bisabolene are enzyme dependent but the formation of gamma-bisabolol in the reaction mixtures was a Mg2+ dependent solvolysis of nerolidyl diphosphate. Enzyme mechanisms are proposed for the formation of delta-cadinene from (E,E)-farnesyl diphosphate and for the formation of delta-cadinene, E-(beta)- farnesene and beta-bisabolene from (3R)-nerolidyl diphosphate.