title: 4-Coumaroyl and caffeoyl shikimic acids inhibit 4-coumaric acid:coenzyme A ligases and modulate metabolic flux for 3-hydroxylation in monolignol biosynthesis of Populus trichocarpa.
abstract: Downregulation of 4-coumaric acid:coenzyme A ligase (4CL) can reduce lignin content in a number of plant species. In lignin precursor (monolignol) biosynthesis during stem wood formation in Populus trichocarpa, two enzymes, Ptr4CL3 and Ptr4CL5, catalyze the coenzyme A (CoA) ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. CoA ligation of 4-coumaric acid is essential for the 3-hydroxylation of 4-coumaroyl shikimic acid. This hydroxylation results from sequential reactions of 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) and 4-coumaric acid 3-hydroxylase 3 (PtrC3H3). Alternatively, 3-hydroxylation of 4-coumaric acid to caffeic acid may occur through an enzyme complex of cinnamic acid 4-hydroxylase 1 and 2 (PtrC4H1 and PtrC4H2) and PtrC3H3. We found that 4-coumaroyl and caffeoyl shikimic acids are inhibitors of Ptr4CL3 and Ptr4CL5. 4-Coumaroyl shikimic acid strongly inhibits the formation of 4-coumaroyl-CoA and caffeoyl-CoA. Caffeoyl shikimic acid inhibits only the formation of 4-coumaroyl-CoA. 4-Coumaroyl and caffeoyl shikimic acids both act as competitive and uncompetitive inhibitors. Metabolic flux in wild-type and PtrC3H3 downregulated P. trichocarpa transgenics has been estimated by absolute protein and metabolite quantification based on liquid chromatography-tandem mass spectrometry, mass action kinetics, and inhibition equations. Inhibition by 4-coumaroyl and caffeoyl shikimic acids may play significant regulatory roles when these inhibitors accumulate.
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Chien-Yuan
authors.initials: CY
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jack P
authors.initials: JP
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China.
authors.forename: Quanzi
authors.initials: Q
authors.lastname: Li
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Hsi-Chuan
authors.initials: HC
authors.lastname: Chen
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Liu
authors.affiliation: W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Philip
authors.initials: P
authors.lastname: Loziuk
authors.affiliation: Department of Electrical and Computer Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Jina
authors.initials: J
authors.lastname: Song
authors.affiliation: Department of Electrical and Computer Engineering, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: Cranos
authors.initials: C
authors.lastname: Williams
authors.affiliation: W.M. Keck FT-ICR Mass Spectrometry Laboratory, Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Muddiman
authors.affiliation: Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: ron_sederoff@ncsu.edu.
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Sederoff
authors.affiliation: State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China; Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC 27695, USA. Electronic address: vchiang@ncsu.edu.
authors.forename: Vincent L
authors.initials: VL
authors.lastname: Chiang
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism / Q000494:pharmacology*; D006900:Hydroxylation / Q000187:drug effects; D010940:Plant Proteins / Q000378:metabolism*; D032107:Populus / Q000187:drug effects* / Q000201:enzymology* / Q000378:metabolism; D011422:Propionates; D012765:Shikimic Acid / Q000494:pharmacology*
chemical_list: C403206:4-hydroxycinnamoyl-CoA; D000214:Acyl Coenzyme A; D003373:Coumaric Acids; D010940:Plant Proteins; D011422:Propionates; C058644:4-coumaroyl-coenzyme A; D012765:Shikimic Acid; D003066:Coenzyme A Ligases; C495469:p-coumaric acid
keywords: 4-coumaroyl and caffeoyl shikimic acids; LC-MS/MS; Populus trichocarpa; metabolic flux; monolignol biosynthesis; reaction and inhibition kinetics