Paiva N L, Sun Y, Dixon R A, VanEtten H D, Hrazdina G
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73402.
Arch Biochem Biophys. 1994 Aug 1;312(2):501-10. doi: 10.1006/abbi.1994.1338.
Isoflavone reductase (IFR) reduces achiral isoflavones to chiral isoflavanones during the biosynthesis of chiral pterocarpan phytoalexins. A cDNA clone for IFR from pea (Pisum sativum) was isolated using the polymerase chain reaction and expressed in Escherichia coli. Analysis of circular dichroism (CD) spectra of the reduction product sophorol obtained using the recombinant enzyme indicated that the isoflavanone possessed the 3R stereochemistry, in contrast to previous reports indicating a 3S-isoflavanone as the product of the pea IFR. Analysis of CD spectra of sophorol produced using enzyme extracts of CuCl2-treated pea seedlings confirmed the 3R stereochemistry. Thus, the stereochemistry of the isoflavanone intermediate in (+)-pisatin biosynthesis in pea is the same as that in (-)-medicarpin biosynthesis in alfalfa, although the final pterocarpans have the opposite stereochemistry. At the amino acid level the pea IFR cDNA was 91.8 and 85.2% identical to the IFRs from alfalfa and chickpea, respectively. IFR appears to be encoded by a single gene in pea. Its transcripts are highly induced in CuCl2-treated seedlings, consistent with the appearance of IFR enzyme activity and pisatin accumulation.
异黄酮还原酶（IFR）在手性紫檀芪植物抗毒素的生物合成过程中，将非手性异黄酮还原为手性异黄烷酮。利用聚合酶链反应从豌豆（Pisum sativum）中分离出IFR的cDNA克隆，并在大肠杆菌中表达。对使用重组酶获得的还原产物槐醇的圆二色性（CD）光谱分析表明，该异黄烷酮具有3R立体化学结构，这与之前报道的豌豆IFR产物为3S-异黄烷酮的结果相反。对用CuCl2处理的豌豆幼苗的酶提取物产生的槐醇的CD光谱分析证实了3R立体化学结构。因此，豌豆中（+）-豌豆素生物合成过程中异黄烷酮中间体的立体化学结构与苜蓿中（-）-苜蓿素生物合成过程中的相同，尽管最终的紫檀芪具有相反的立体化学结构。在氨基酸水平上，豌豆IFR cDNA与苜蓿和鹰嘴豆的IFR分别有91.8%和85.2%的同一性。IFR似乎由豌豆中的单个基因编码。其转录本在经CuCl2处理的幼苗中高度诱导，这与IFR酶活性的出现和豌豆素积累一致。
