New routes for lignin biosynthesis defined by biochemical characterization of recombinant ferulate 5-hydroxylase, a multifunctional cytochrome P450-dependent monooxygenase.
The enzymes and genes of the lignin biosynthetic pathway have been studied for several decades, but the gene encoding ferulate 5-hydroxylase (F5H) was cloned only 3 years ago by T-DNA tagging in Arabidopsis. To characterize the enzyme in detail, we have expressed F5H in yeast. According to current models of the phenylpropanoid pathway, F5H catalyzes the hydroxylation of ferulate to 5-hydroxyferulate; however, our studies indicate that the enzyme also uses coniferaldehyde and coniferyl alcohol as substrates. Unexpectedly, the K(m) values measured for the latter two substrates are three orders of magnitude lower than that measured for ferulic acid, suggesting that in lignifying tissues, syringyl monomers may be derived from their guaiacyl counterparts by hydroxylation and subsequent methylation. Thus, F5H may function later in the lignin biosynthetic pathway than was originally proposed. To further test this model, recombinant F5H was incubated together with ferulic acid, coniferaldehyde, or coniferyl alcohol in the presence of native or recombinant Arabidopsis caffeic acid/5-hydroxyferulic acid O-methyltransferase and [(14)C]S-adenosylmethionine. In all cases, the corresponding radiolabeled sinapyl derivatives were synthesized, indicating that the necessary enzymes required for this pathway are present in Arabidopsis. Taken together, these data suggest that the previously accepted pathway for lignin biosynthesis is likely to be incorrect.
Humphreys
J M
JM
Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153, USA.
Hemm
M R
MR
Chapple
C
C
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins; D003001:Cloning, Molecular / Q000379:methods; D003577:Cytochrome P-450 Enzyme System; D004926:Escherichia coli; D006900:Hydroxylation; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis*; D008745:Methylation; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D008031:Lignin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C097486:CYP84A1 protein, Arabidopsis