Coniferyl aldehyde 5-hydroxylation and methylation direct syringyl lignin biosynthesis in angiosperms.
A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H/COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H/COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k(cat)/K(m) of CAld5H for coniferyl aldehyde was approximately 140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K(i) = 0.59 microM) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H/COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.
Osakabe
K
K
Plant Biotechnology Research Center, School of Forestry and Wood Products, Michigan Technological University, 1400 Townsend Drive, Houghton, MI 49931, USA.
Tsao
C C
CC
Li
L
L
Popko
J L
JL
Umezawa
T
T
Carraway
D T
DT
Smeltzer
R H
RH
Joshi
C P
CP
Chiang
V L
VL
D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary; D006900:Hydroxylation; D007700:Kinetics; D008031:Lignin / Q000096:biosynthesis*; D019684:Magnoliopsida / Q000201:enzymology*; D008745:Methylation; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014197:Trees / Q000201:enzymology
D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D008031:Lignin; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C120927:Ald5H protein, Liquidambar styraciflua; D008780:Methyltransferases; C019608:caffeate O-methyltransferase