Abstract
Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and thus is the first committed step in PA biosynthesis. The enzyme was purified to near homogeneity from PA-rich leaves of the legume Desmodium uncinatum (Jacq.) DC, partially sequenced and the corresponding cDNA cloned. The identity of the enzyme was confirmed by expressing active recombinant LAR in Escherichia coli and in tobacco and white clover. The enzyme is a monomer of 43 kDa (382 amino acids) and is most active synthesizing catechin (specific activity of approximately 10 micromol min+1 mg of protein+1) but also synthesizes afzelechin and gallocatechin. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Unlike all other plant isoflavone reductase homologues that are about 320 amino acids long, LAR has an additional 65-amino acid C-terminal extension whose function is not known. Curiously, although Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome. This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/epicatechin-based PA similar to many other plants.
Highlights
Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and is the first committed step in PA biosynthesis
Polymerization continues by sequential addition of flavan-3,4-diols to the growing chain. This makes the pathway unusual because during the synthesis of a polymer of ten units only 10% of the monomer flux need proceed the full-length of the pathway while the 90% contributing to extension units does not pass through the catechin or epicatechin steps (Fig. 1.) The only biochemical steps that have been demonstrated in vitro are the NADPH-dependent reduction of (ϩ)3,4cis-leucocyanidin to catechin catalyzed by leucoanthocyanidin reductase (LAR) that removes the C4 hydroxyl group (Fig. 1) and an anthocyanidin reductase that converts anthocyanidin to epicatechin [4]
The elution step utilized the extraordinary affinity of LAR for NADPH (ϳ0.4 M) to help separate LAR from many other enzymes that bind to the dye column via their NADPH binding site but with lower affinity for NADPH
Summary
PURIFICATION OF LEGUME LEUCOANTHOCYANIDIN REDUCTASE AND MOLECULAR CLONING OF ITS cDNA*□S. Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin, an initiating monomer of condensed tannin or proanthocyanidin (PA) synthesis, from 3,4-cis-leucocyanidin and is the first committed step in PA biosynthesis. LAR is most closely related to the isoflavone reductase group of plant enzymes that are part of the Reductase-Epimerase-Dehydrogenase (RED) family of proteins. Arabidopsis makes PA, there is no obvious LAR orthologue in the Arabidopsis genome This may be because Arabidopsis seems to produce only an epicatechin, rather than a dual catechin/ epicatechin-based PA similar to many other plants. PCBER, phenylcoumaran-benzylic ether reductase; RED, ReductaseEpimerase-Dehydrogenase; SPP, sucrose phosphate phosphatase; TDS, tannin-deficient seed; TT, transparent testa.
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