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 over (+) 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.
DiCenzo
Gregory L
GL
Division of Plant Pathology and Microbiology, Plant Science Department, College of Agriculture, University of Arizona, P.O. Box 210036, Tucson, AZ 85721-0036, USA.
VanEtten
Hans D
HD
D000595:Amino Acid Sequence; D000981:Antiprotozoal Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D018548:Cotyledon / Q000737:chemistry / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D007529:Isoflavones / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D018532:Pisum sativum / Q000235:genetics / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism*; D036343:Pterocarpans / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013997:Time Factors; D000094162:Phytoalexins
D000981:Antiprotozoal Agents; D018076:DNA, Complementary; D007529:Isoflavones; D009249:NADP; D010088:Oxidoreductases; D010936:Plant Extracts; D036343:Pterocarpans; C065753:isoflavanone; C088468:sophorol; C001449:inermin; D000094162:Phytoalexins; C003161:pisatin