(+)-Larreatricin hydroxylase, an enantio-specific polyphenol oxidase from the creosote bush (Larrea tridentata)

Abstract

An enantio-specific polyphenol oxidase (PPO) was purified approximately 1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO ( approximately 43 kDa) has a central role in the biosynthesis of the creosote bush 8-8' linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3'-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (-)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.

Fig. 1.

Proposed biosynthetic pathway to creosote bush (L. tridentata) lignans.

Scheme 1.

Fig. 2.

Enantio-specificity of (+)-larreatricin 3′-hydroxylase. (A) Time course of larreatricin (2) substrate depletion catalyzed by (+)-larreatricin 3′-hydroxylase. ▪, (+)-larreatricin; •, (–)-larreatricin. (B) Chiral separation of synthetic (+)- and (–)-larreatricins (2). (C) Enantiomeric composition of larreatricin (2) after incubation for 4 h with larreatricin 3′-hydroxylase. (D) Chiral separation of synthetic (+)- and (–)-3′-hydroxylarreatricins (6). (E) Enantiomeric composition of enzymatically formed 3′-hydroxylarreatricin (6). S, solvent peak.

Fig. 3.

Alignment of deduced amino acid sequences of cDNA encoding (+)-larreatricin 3′-hydroxylase (LtLH) and those of selected plant PPOs (, –33). Amino acid sequences of peptides derived from purified enzyme are underlined in red. Identical and similar amino acid residues are shown in black and gray, respectively. Copper binding domains A and B are indicated by dark blue and light blue boxes, respectively. Predicted N-terminal and C-terminal processing sites are indicated by arrows.