Crystal Structures of Pinoresinol-Lariciresinol and Phenylcoumaran Benzylic Ether Reductases and Their Relationship to Isoflavone Reductases

Tongpil Min,Hiroyuki Kasahara,Diana L Bedgar,Buhyun Youn,Paulraj K Lawrence,David R Gang,Steven C Halls,Hajeung Park,Jacqueline L Hilsenbeck,Laurence B Davin,Norman G Lewis,Chulhee Kang

doi:10.1074/jbc.m308493200

Abstract

Despite the importance of plant lignans and isoflavonoids in human health protection (e.g. for both treatment and prevention of onset of various cancers) as well as in plant biology (e.g. in defense functions and in heartwood development), systematic studies on the enzymes involved in their biosynthesis have only recently begun. In this investigation, three NADPH-dependent aromatic alcohol reductases were comprehensively studied, namely pinoresinol-lariciresinol reductase (PLR), phenylcoumaran benzylic ether reductase (PCBER), and isoflavone reductase (IFR), which are involved in central steps to the various important bioactive lignans and isoflavonoids. Of particular interest was in determining how differing regio- and enantiospecificities are achieved with the different enzymes, despite each apparently going through similar enone intermediates. Initially, the three-dimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 A resolutions, respectively. Not only do they share high gene sequence similarity, but their structures are similar, having a continuous alpha/beta NADPH-binding domain and a smaller substrate-binding domain. IFR (whose crystal structure is not yet obtained) was also compared (modeled) with PLR and PCBER and was deduced to have the same overall basic structure. The basis for the distinct enantio-specific and regio-specific reactions of PCBER, PLR, and IFR, as well as the reaction mechanism and participating residues involved (as identified by site-directed mutagenesis), are discussed.

Highlights

Vascular plants produce a wide variety of plant defense and health-protecting metabolites, such as the lignans and isoflavonoids, with both classes of compounds being offshoots of the phenylpropanoid pathway [1, 2]
Overall Structures—The asymmetric units of crystalline PCBER_Pt1 and PLR_Tp1 have two and four independent molecules, respectively, which are virtually superimposable in both cases with a root mean square deviation of 0.87 Å between two PCBER_Pt1 molecules and 1.0 Å among four PLR_Tp1 molecules
The overall gross structures of both crystalline PLR_Tp1 and PCBER_Pt1 are composed of two domains (N- and C-terminal) connected by five separate peptide segments, with the two domains forming a cleft between them (Fig. 2, A and B)

Summary

Introduction

Vascular plants produce a wide variety of plant defense and health-protecting metabolites, such as the (oligomeric) lignans and isoflavonoids, with both classes of compounds being offshoots of the phenylpropanoid pathway [1, 2]. The threedimensional x-ray crystal structures of both PLR_Tp1 and PCBER_Pt1 were solved and refined to 2.5 and 2.2 Å resolutions, respectively, the structures of which allow incisive comparative studies to be made between the enantiospecific and regiospecific reactions of PCBER_Pt1, PLR_Tp1, and IFR.

Results

Conclusion