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• 2035-15-6 (-)-maackiain 

Relevant academic research and scientific papers

Studies on the late steps of (+) pisatin biosynthesis: Evidence for (-) enantiomeric intermediates

Pisatin, a 6a-hydroxyl-pterocarpan phytoalexin from pea (Pisum sativum L.), is relatively unique among naturally occurring pterocarpans by virtue of the (+) stereochemistry of its 6a-11a C-C bond. However, pisatin synthesizing pea tissue has an isoflavone reductase, first identified in alfalfa, which acts on the (-) antipode. In order to establish the natural biosynthetic pathway to (+) pisatin, and to evaluate the possible involvement of intermediates with a (-) chirality in its biosynthesis, we administered chiral, tritium-labeled, isoflavanones and pterocarpans to pisatin-synthesizing pea cotyledons and compared the efficiency of their incorporation. Pea incorporated the isoflavanone, (-) sophorol, more efficiently than either its (+) antipode, or the pterocarpans (+) or (-) maackiain. (-) Sophorol was also metabolized by protein extracts from pisatin-synthesizing pea seedlings in a NADPH-dependent manner. Three products were produced. One was the isoflavene (7,2′-dihydroxy-4′,5′-methylenedioxyisoflav-3-ene), and another had properties consistent with the isoflavanol (7,2′-dihydroxy- 4′,5′-methylenedioxyisoflavanol), the expected product for an isoflavanone reductase. A cDNA encoding sophorol reductase was also isolated from a cDNA library made from pisatin-synthesizing pea. The cloned recombinant sophorol reductase preferred (-) sophorol(+) sophorol as a substrate and produced 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol. Although no other intermediates in (+) pisatin biosynthesis were identified, the results lend additional support to the involvement of intermediates of (-) chirality in (+) pisatin synthesis.

Inactivation of pea genes by RNAi supports the involvement of two similar O-methyltransferases in the biosynthesis of (+)-pisatin and of chiral intermediates with a configuration opposite that found in (+)-pisatin

(+)-Pisatin, the major phytoalexin of pea (Pisum sativum L.), is believed to be synthesized via two chiral intermediates, (-)-7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanone [(-)-sophorol] and (-)-7,2′-dihydroxy-4′,5′-methylenedioxyisoflavanol [(-)-DMDI]; both have an opposite C-3 absolute configuration to that found at C-6a in (+)-pisatin. The expression of isoflavone reductase (IFR), which converts 7,2′-dihydroxy-4′,5′-methylenedioxyisoflavone (DMD) to (-)-sophorol, sophorol reductase (SOR), which converts (-)-sophorol to (-)-DMDI, and hydroxymaackiain-3-O-methyltransferase (HMM), believed to be the last step of (+)-pisatin biosynthesis, were inactivated by RNA-mediated genetic interference (RNAi) in pea hairy roots. Some hairy root lines containing RNAi constructs of IFR and SOR accumulated DMD or (-)-sophorol, respectively, and were deficient in (+)-pisatin biosynthesis supporting the involvement of chiral intermediates with a configuration opposite to that found in (+)-pisatin in the biosynthesis of (+)-pisatin. Pea proteins also converted (-)-DMDI to an achiral isoflavene suggesting that an isoflavene might be the intermediate through which the configuration is changed to that found in (+)-pisatin. Hairy roots containing RNAi constructs of HMM also were deficient in (+)-pisatin biosynthesis, but did not accumulate (+)-6a-hydroxymaackiain, the proposed precursor to (+)-pisatin. Instead, 2,7,4′-trihydroxyisoflavanone (TIF), daidzein, isoformononetin, and liquiritigenin accumulated. HMM has a high amino acid similarity to hydroxyisoflavanone-4′-O-methyltransferase (HI4′OMT), an enzyme that methylates TIF, an early intermediate in the isoflavonoid pathway. The accumulation of these four compounds is consistent with the blockage of the synthesis of (+)-pisatin at the HI4′OMT catalyzed step resulting in the accumulation of liquiritigenin and TIF and the diversion of the pathway to produce daidzein and isoformononetin, compounds not normally made by pea. Previous results have identified two highly similar "HMMs" in pea. The current results suggest that both of these O-methyltransferases are involved in (+)-pisatin biosynthesis and that one functions early in the pathway as HI4′OMT and the second acts at the terminal step of the pathway.