Enzymic Synthesis of Lignin Precursors Comparison of Cinnamoyl‐CoA Reductase and Cinnamyl Alcohol: NADP⁺ Dehydrogenase from Spruce (Picea abies L.) and Soybean (Glycine max L.)

Abstract

Cambial sap of spruce (Picea abies) proved to be a good source for isolation of cinnamoyl-CoA reductasc and cinnamyl alcohol :NADP+ dehydrogenase. Apparently homogeneous enzymes were obtained by a multistep procedure including dye-ligand chromatography and for the reductase also affinity chromatography on (coenzymc A)-agarose. An improved purification procedure for the reductase from soybean cell cultures is also reported.Molecular weights and subunit composition of reductase and dehydrogenase from spruce are very similar to those of the corresponding enzymes from soybean.Reduction of feruloyl-CoA to coniferaldehyde catalysed by the reductase is a freely reversible reaction with an equilibrium constant of 5.6 x M at pH 6.25. A strong dependence of the Michaelis constants on the type of buffer was found. For reductase the K,-value of feruloyl-CoA in phosphate buffer (5.2 pM) is about 14-times smaller than in citrate buffer (73 pM).Pronounced differences in substrate specificities between the enzymes from spruce and soybean were found, which reflect the different lignin composition of gymnosperms and dicotyledenous angiosperms. From the kinetic constants of the enzymes it can be concluded that under physiological conditions feruloyl-CoA is the preferred substrate for the reductase from both sources whereas sinapoyl-CoA is a substrate only for the soybean reductase and sinapyldehyde a substrate only for the soybean dehydrogenase.4-Coumaroyl-CoA is a poor substrate for the reductase from both spruce and soybean.This result is consistent with the low content of 4-coumaryl alcohol units in gymnosperm and angiosperm lignin.

Purification and Characterization of Cinnamyl Alcohol Dehydrogenase Isoforms from the Periderm of Eucalyptus gunnii Hook

“…Comparison of the kinetic properties of the HMW homodimer and the heterodimer reveals clear differences in the affinity of the different forms for their in vivo substrates (hydroxycinnamaldehydes), which, as suggested by Luderitz and Grisebach (1981) in the case of spruce, may be of physiological significance with regard to the synthesis of different lignin types.…”

“…CAD purified from poplar (Savidge, 1989) is reported to be a homodimer (40-kD subunit), as is the CAD from spruce (Luderitz and Grisebach, 1981) (41.7-kD subunit) and loblolly pine (O'Malley et al, 1992) (44-kD subunit), whereas the CAD purified from tobacco (Halpin et al, 1992) appears to be a heterodimer (44-and 42.5-kD subunits). The observation that Eucalyptus periderm CAD 2P exists as a homodimer as well as a heterodimer was unexpected, since Eucalyptus xylem CAD has been reported to exist as a heterodimer (Goffner et al, 1992).…”

“…CAD is one of the two branch enzymes of general phenylpropanoid metabolism specific to hydroxycinnamyl alcohol (monolignol) synthesis catalyzing the conversion of hydroxycinnamaldehydes to the corresponding alcohols (Grisebach, 1981). Monolignols are the monomeric precursors of the complex phenolic polymer lignin that, after cellulose, constitutes the most abundant biopolymer on Earth and accounts for up to 30% dry weight of secondary xylem in woody species.…”

Variation in Lignin Content and Composition (Mechanisms of Control and Implications for the Genetic Improvement of Plants)

“…Conifer CCR and CAD have higher affinity for substrates that are precursors to guaiacyl lignins (feruloyl-CoA and coniferaldehyde) than they do for the syringyl-precursor substrates (sinapoyl-COA and sinapaldehyde) (Liideritz and Grisebach, 1981; OMalley et al, 1992;Galliano et al, 1993). In spruce, the maximal reaction rates for both CCR and CAD with the syringyl precursors were 10% of the rates with the guaiacyl precursors.…”

“…Sinapate 4CL activity is rare even in angiosperms, despite the fact that they produce syringyl lignins (Kutsuki et al, 198213). Paradoxically, E. cristagalli catalyzes the conversion of sinapate to sinapoyl-COA even though the vast majority of syringyl lignin-producing angiosperms do not.Conifer CCR and CAD have higher affinity for substrates that are precursors to guaiacyl lignins (feruloyl-CoA and coniferaldehyde) than they do for the syringyl-precursor substrates (sinapoyl-COA and sinapaldehyde) (Liideritz and Grisebach, 1981; OMalley et al, 1992;Galliano et al, 1993). In spruce, the maximal reaction rates for both CCR and CAD with the syringyl precursors were 10% of the rates with the guaiacyl precursors.…”