Two O-methyltransferases in lignin monomer biosynthetic pathway: Caffeic acid O-methyltransferase and caffeoyl-CoA O-methyltransferase

Two O-methyltransferases (caffeic acid O-methyltransferase and caffeoyl-CoA O-methyltransferase) involved in lignin monomer biosynthetic pathway were studied. Aspen caffeic acid O-methyltransferase (CAOMT) was recombinantly expressed in Escherichia coli using pET-23 vector and purified to homogeneity by Phenyl-Sepharose and S-adenosylhomocysteine-agarose chromatographies. CAOMT has a subunit of 40 kDa and native gradient gel electrophoresis indicated the active form is a dimer. Substrate specificity of CAOMT was investigated using over 20 phenolic compounds, which define the nature of the substrate binding site and required substrate characteristics. The kinetics of CAOMT was studied and an ordered Bi Bi sequential mechanism with two non-produetive complexes model was proposed. The role of thiol side chain(s) in the catalytic mechanism was investigated since CAOMT was inhibited by p-chloromercuribenzoate (p-CMB). Site-directed mutagenesis was performed to mutate cysteine-276 and cysteine-283 because they are highly conserved among higher plant CAOMTs. Results showed CAOMT mutants were enzymatically active and still sensitive to p-CMB, which indicated thiol(s) were not important in the catalytic mechanism of CAOMT and the sensitivity to thiol inhibitor was probably due to steric hindrance caused by the reaction of cysteine thiol(s) with thiol inhibitor. Regarding to caffeoyl-CoA O-methyltransferase (CCoAOMT), a cDNA encoding this enzyme was isolated from aspen developing secondary xylem. Sequencing alignment with CCoAOMTs from other plants showed high similarity and Southern analysis indicated there is only one copy of CCoAOMT gene in aspen genome. Northern analysis showed CCoAOMT was tissue specifically expressed in xylem but not in bark or leaves. Seasonal expression of CCoAOMT and CAOMT were examined comparatively and a biphasic expression pattern was found for both OMTs, which indicated both OMTs are involved in lignin monomer biosynthetic pathway. Further, CCoAOMT was recombinantly expressed in E. coli and its substrate specificity was examined. CCoAOMT showed activity with both caffeoyl-CoA and 5-hydroxyferuloy-CoA at a ratio of 2.5 to 1. In contrast, aspen CAOMT, which is active on both caffeic acid and 5-hydroxyferulic acid, showed a ratio of 1 to 2.2. The preference of CCoAOMT for caffeoyl-CoA and the preference of CAOMT for 5-hydroxyferulate may suggest the different role of each OMT plays in lignin monomer biosynthesis. To assist with the study ofcaffeoyl-CoA OMT, p-hydroxycinnamate: CoA ligase was isolated from aspen xylem and used to synthesis of caffeoyl-CoA. This enzymatic synthesis method, which yielded 40% product, is rapid and simple and provides a cost effective alternative to the use of commerciai acyl CoA ligase.