Structure and function of enzymes involved in the biosynthesis of phenylpropanoids

Abstract

As a major component of plant specialized metabolism, phenylpropanoid biosynthetic pathways provide anthocyanins for pigmentation, flavonoids such as flavones for protection against UV photodamage, various flavonoid and isoflavonoid inducers of Rhizobium nodulation genes, polymeric lignin for structural support and assorted antimicrobial phytoalexins. As constituents of plant-rich diets and an assortment of herbal medicinal agents, the phenylpropanoids exhibit measurable cancer chemopreventive, antimitotic, estrogenic, antimalarial, antioxidant and antiasthmatic activities. The health benefits of consuming red wine, which contains significant amounts of 3,4',5-trihydroxystilbene (resveratrol) and other phenylpropanoids, highlight the increasing awareness in the medical community and the public at large as to the potential dietary importance of these plant derived compounds. As recently as a decade ago, little was known about the three-dimensional structure of the enzymes involved in these highly branched biosynthetic pathways. Ten years ago, we initiated X-ray crystallographic analyses of key enzymes of this pathway, complemented by biochemical and enzyme engineering studies. We first investigated chalcone synthase (CHS), the entry point of the flavonoid pathway, and its close relative stilbene synthase (STS). Work soon followed on the O-methyl transferases (OMTs) involved in modifications of chalcone, isoflavonoids and metabolic precursors of lignin. More recently, our groups and others have extended the range of phenylpropanoid pathway structural investigations to include the upstream enzymes responsible for the initial recruitment of phenylalanine and tyrosine, as well as a number of reductases, acyltransferases and ancillary tailoring enzymes of phenylpropanoid-derived metabolites. These structure-function studies collectively provide a comprehensive view of an important aspect of phenylpropanoid metabolism. More specifically, these atomic resolution insights into the architecture and mechanistic underpinnings of phenylpropanoid metabolizing enzymes contribute to our understanding of the emergence and on-going evolution of specialized phenylpropanoid products, and underscore the molecular basis of metabolic biodiversity at the chemical level. Finally, the detailed knowledge of the structure, function and evolution of these enzymes of specialized metabolism provide a set of experimental templates for the enzyme and metabolic engineering of production platforms for diverse novel compounds with desirable dietary and medicinal properties.

Fig. 1

Phenylpropanoid biosynthetic pathways. Enzymes and representative reactions of each major pathway branch are shown. In all panels, capital letters marked with asterisks designate corresponding panels of Fig. 2 for structurally characterized pathway enzymes, and color indicates highly represented enzyme classes: cytochrome P450-catalyzed reactions are highlighted in red, short-chain dehydrogenase/reductase (SDR) reactions are green, and O-methyltransferase (OMT) reactions are lavender. (A) The general phenylpropanoid pathway leading from phenylalanine to p-coumaroyl-CoA, the entry point to each major downstream pathways. Additional offshoot product classes are indicated. (B) Monolignol biosynthetic grid leading to polymeric lignin, oligomeric lignans, and monomeric phenylpropenes. Newly discovered ‘third dimension’ shown in blue. Monolignol end products are in shadowed boxes. (C) Structurally characterized lignan reductase reactions. (D) Flavonoid pathway leading to anthocyanin pigments, isoflavonoid ‘phytoestrogens’, polymeric phlobaphenes and condensed tannins, and various antimicrobial phytoalexins. Related biosynthesis of resveratrol is also shown.

Fig. 1

Phenylpropanoid biosynthetic pathways. Enzymes and representative reactions of each major pathway branch are shown. In all panels, capital letters marked with asterisks designate corresponding panels of Fig. 2 for structurally characterized pathway enzymes, and color indicates highly represented enzyme classes: cytochrome P450-catalyzed reactions are highlighted in red, short-chain dehydrogenase/reductase (SDR) reactions are green, and O-methyltransferase (OMT) reactions are lavender. (A) The general phenylpropanoid pathway leading from phenylalanine to p-coumaroyl-CoA, the entry point to each major downstream pathways. Additional offshoot product classes are indicated. (B) Monolignol biosynthetic grid leading to polymeric lignin, oligomeric lignans, and monomeric phenylpropenes. Newly discovered ‘third dimension’ shown in blue. Monolignol end products are in shadowed boxes. (C) Structurally characterized lignan reductase reactions. (D) Flavonoid pathway leading to anthocyanin pigments, isoflavonoid ‘phytoestrogens’, polymeric phlobaphenes and condensed tannins, and various antimicrobial phytoalexins. Related biosynthesis of resveratrol is also shown.

Fig. 2

Elucidated structures of the phenylpropanoid pathways. Crystal structures of: (A) PAL. (B) COMT in complex with SAH and ferulic acid. (C) CCoAOMT in complex with sinapoyl-CoA. (D) SAD in complex with NADP+. (E) CAD in complex with NADP+. (F) EGS in complex with NADP+ and (7S,8S )-ethyl (7,8-methylene)-dihydroferulate. (G) PLR. (H) PCBER. (I) CHI in complex with naringenin. (J) CHR in complex with NADP+. (K) CHS in complex with malonyl-CoA. (L) STS in complex with resveratrol. (M) DFR in complex with NADP and dihydroquercetin. (N) ANS in complex with naringenin. (O) ANR. (P) IFR. (Q) VR. (R) IOMT in complex with SAH and 4′-hydroxy-7-methoxyisoflavone. (S) Dm3MAT3 in complex with malonyl-CoA. (T) UGT71G1 in complex with UDP-glucose. (U) VvGT1 in complex with UDP-2FGlc and kaempferol. This figure was produced with PyMOL (http://www.pymol.org).

Fig. 3

Structural comparisons within the same families. (A) Comparison of the overall structure of COMT and CCoAOMT monomers (colored in cyan and orange respectively). SAH and ferulic acid are represented as yellow sticks. (B) Comparison of the overall structure of stilbene synthase (STS: monomers in orange and magenta) and chalcone synthase in complex with malonyl CoA (CHS: monomers in cyan and green; malonyl-CoA as yellow sticks). (C) Comparison of the overall structure of cinnamyl/sinapyl alcohol dehydrogenase (CAD: monomers in orange and magenta) and sinapyl alcohol dehydrogenases (SAD: monomers in cyan and green). (D) Comparison of the active sites of CAD and SAD (same colors as panel C). Residues are labeled according to CAD sequence. NADP+ is represented as yellow sticks. (E) Comparison of the different reductases of the phenylpropanoid pathway structurally characterized (PCBER is represented in green, PLR in cyan, DFR in orange, IFR in magenta and VR in blue). (F) Comparison of glycosyltransferases of the phenylpropanoid (UGT85H2 in green, UGT71G1 in cyan and VvGT1 in orange). UDP-2FGlc and kaempferol from VvGT1 structure are represented as yellow sticks. This figure was produced with PyMOL (http://www.pymol.org).