As shown in Figure 7.1, the pathway to the monolignols 3-5 includes two methylation steps. These have been shown to be catalyzed by caffeic acid O-methyltransferases (COMTs) and caffeoyl CoA O-methyltransferases (CCOMTs). COMT activity was first detected in cambial tissue isolated from an apple tree species by Finkle and Nelson (134). It was thought for much time that COMTs could catalyze the formation of both ferulic (11) and sinapic (13) acids from caffeic (10) and 5-hydroxyferulic (12) acids, respectively. However, in 1989, Pakusch et al. (135) described an enzyme, CCOMT, capable of converting caffeoyl CoA (15) into feruloyl CoA (16). Additionally, downregulation of the COMT gene later unambiguously

NADPH

Figure 7.9 (A) Structure of the substrate-binding pocket of NADP+ binary form of AtCAD5 showing the catalytic Zn2+ ion (red sphere) tetrahedrally coordinated by Cys47, His69, Cys163 and Glu70 (blue). The NADP+ molecule (orange) is held by Val192, Ser211, Ser212, Ser213, Lys216 and Gly275 (green) (133). [Possible hydrogen bonds are shown as black dotted lines.] (B) Proposed proton shuttling mechanism during the reduction process in the active site of the AtCAD5. Solid arrows indicate the movement of two electrons among the functional groups during substrate reduction (133). The possible hydrogen bonds involved are shown with dotted lines. (Reprinted from Organic and Biomolecular Chemistry, vol. 4, Youn, B., Camacho, R., Moinuddin, S. G.A., Lee, C., Davin, L. B., Lewis, N. G. & Kang, C., Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4, pp. 1687-1697, Copyright 2006, with permission from The Royal Society of Chemistry.) (Reproduced in color as Plate 20.)
established its role in syringyl (S) unit formation and not the initial step involving G-lignin deposition (136). Genes encoding COMT and CCOMT were first reported in 1991 by Bugos etal. (137)/Gowri etal. (138) and by Schmitt etal. (139), respectively, with the actual biochemical/biophysical function for COMT being determined later by Atanassova et al. (136).

In the TAIR database, there are 17 genes putatively annotated as COMTs and five as CCOMTs. Investigations in our laboratory have indicated that out of the 17 COMTs only one, AtCOMT1, was capable of methylating caffeic (10)/5-OH ferulic (12) acids, caffeyl (20)/5-hydroxyconiferyl (22) aldehydes and caffeyl (2)/5-hydroxyconiferyl (4) alcohols in vitro. The efficiency of the reaction is higher when the aldehydes 20 and 22 are used as substrates (Zhang etal., manuscript in preparation). Only two CCOMTs, AtCCOMT1 and AtCCOMT2, have been investigated: caffeoyl (15) and 5-hydroxyferuloyl (17) CoAs are the preferred substrates for AtCCOMT1, with 10 and 12 not being converted into ferulic (11) and sinapic (13) acids, respectively. AtCCOMT2 more efficiently methylated quercetin (Takahashi etal., manuscript in preparation).