7.3.8.1 CAD1

In addition to the bona fide CADs [such as from tobacco (126), loblolly pine (35) and Arabidopsis (56, 57)], there continue to be a number of reports proposing that another class of dehydrogenases (so-called CAD1) is involved in monolignol/lignin biosynthesis (140, 141). These enzymes, by contrast, bear little homology to bona fide CADs and lack both catalytic and structural zinc metal ions (56). For example, a dehydrogenase, annotated as CAD1, was purified from Eucalyptus gunnii in parallel with a bona fide CAD, then called CAD2 (142). CAD1 was shown to have lower affinity for p-coumaryl (19)/coniferyl (21) aldehydes (Km = 70 and 25 ^M) as compared to CAD2 (Km = 1.2 and 1.7 ^M), with sinapyl aldehyde (23) not serving as a substrate; CAD1, however, was not characterized further. It was also presumed to be a monomer (~38 kDa), as compared to CAD2, which is a dimer (~83 kDa) (142). However, the encoding gene (CAD1-5) (140) had only 30/15% similarity/identity to bona fide CADs [e. g., AtCAD5 (56)].

Since then, two other alcohol dehydrogenase genes (so-called NtCAD1-1 and NtCAD1-7) present in tobacco were also obtained by screening a cDNA library with the E. gunnii CAD1-5 coding sequence. Curiously, these were reported as a new step for the biochemical formation of coniferyl alcohol (3) (141). Both dehydrogenases again lacked the Zn catalytic center and the Zn-binding signature found in bona fide CADs, and also had ~30% similarity and ~ 15% identity compared to AtCAD5. The kinetic parameters of both NtCAD1-1 and NtCAD1- 7 were nevertheless investigated using coniferyl aldehyde (21)/alcohol (3) and sinapyl alcohol (5) as potential substrates. Both proteins were apparently capable of catalyzing the forward reaction of reduction of coniferyl aldehyde (21), as well as the oxidation of coniferyl alcohol (3), but they were apparently unable to convert sinapyl alcohol (5) to sinapyl aldehyde (23); the forward reaction was not examined. The Kenz values for both

NtCAD1-1 and NtCAD1-7 were very modest (low) relative to bona fide CADs, i. e., 3230 and 7650 M-1 s-1 versus 348 000 M-1 s-1 for AtCAD5. We would suggest that this family of dehydrogenases, even though they may be able to inefficiently reduce coniferyl alde­hyde (21) to coniferyl alcohol (3) in vitro, have biochemical functions unrelated to that of monolignol/lignin biogenesis. Indeed, as discussed below, a recent study has also reported that genes highly homologous to “CAD1” are instead 2-phenylacetaldehyde reductases. As a further caution, numerous dehydrogenases are known to exhibit broad substrate versatility of varying levels of efficacy.