| Flax seed (Linum usitatissimum) is the richest natural source of the plant lignan 2,3-bis (3-methoxy-4-hydroxybenzyl) butane-1,4-diglucoside, commonly known as secoisolariciresinol diglucoside (SDG). Over the last twenty years or so, a considerable body of epidemiological and tumor inhibitory data has established this natural product as a potent chemo-preventive agent against the onset of breast, prostate, and colon cancers. Understanding the biochemical pathway to this important plant natural product as a means for either increasing its amount in flax seed, or incorporating the pathway into staple dietary foodstuffs (such as wheat or rice) is the paramount goal of this investigation. Empirically, each of the steps in the pathway was investigated through both phytochemical analysis, molecular biology, and biochemical approaches. In this context, radioisotope precursor/tracer studies employing [14C] and [13C] phenylalanine, [14C] coniferin, and [3H] coniferyl alcohol to size segregated developing flax seed capsules, followed by HPLC, radiochemical, ESI-MS, 1H and 13C NMR analysis of purified flax seed monolignols and lignans, yielded important structural information of phenylpropanoid and lignan intermediates as well as subsequent SDG products creating the SDG-HMG ester-linked oligomer(s). In conjunction with this, in vitro assays were conducted on crude flax seed microsome preparations with [3H] coniferyl alcohol, resulting in the formation of (-)-pinoresinol, and on crude flax seed soluble proteins extracts with [14C] pinoresinol, resulting in formation of both (+)-lariciresinol and (+)-secoisolariciresinol. Additionally, genes encoding the protein affording (+)-pinoresinol (so called dirigent protein), and that engendering formation of (-)-lariciresinol (a pinoresinol reductase) were cloned from developing flax seed as well as their respective promoter sequences. The stereo- and regioselective properties of these proteins were assessed after functional recombinant protein expression. Furthermore, partial purification of a soluble ∼40 kDa UDPG: (+)-secoisolariciresinol diglucosyl transferase (SDGT) was conducted. Evaluation of results acquired from this three-point approach has significantly supplemented our understanding of flax seed lignan biosynthesis, revealing both the biosynthetic steps involved in formation the SDG-HMG ester-linked polymer, as well as evidence that two distinct lignan biosynthetic pathways exist in flax seed. |
