title: Functional characterization of two acyltransferases from Populus trichocarpa capable of synthesizing benzyl benzoate and salicyl benzoate, potential intermediates in salicinoid phenolic glycoside biosynthesis.
abstract: Salicinoids are phenolic glycosides (PGs) characteristic of the Salicaceae and are known defenses against insect herbivory. Common examples are salicin, salicortin, tremuloidin, and tremulacin, which accumulate to high concentrations in the leaves and bark of willows and poplars. Although their biosynthetic pathway is not known, recent work has suggested that benzyl benzoate may be a potential biosynthetic intermediate. Two candidate genes, named PtACT47 and PtACT49, encoding BAHD-type acyl transferases were identified and are predicted to produce such benzylated secondary metabolites. Herein described are the cDNA cloning, heterologous expression and in vitro functional characterization of these two BAHD acyltransferases. Recombinant PtACT47 exhibited low substrate selectivity and could utilize acetyl-CoA, benzoyl-CoA, and cinnamoyl-CoA as acyl donors with a variety of alcohols as acyl acceptors. This enzyme showed the greatest Km/Kcat ratio (45.8 nM(-1) s(-1)) and lowest Km values (45.1 μM) with benzoyl-CoA and salicyl alcohol, and was named benzoyl-CoA: salicyl alcohol O-benzoyltransferase (PtSABT). Recombinant PtACT49 utilized a narrower range of substrates, including benzoyl-CoA and acetyl-CoA and a limited number of alcohols. Its highest Km/Kcat (31.8 nM(-1) s(-1)) and lowest Km (55.3 μM) were observed for benzoyl-CoA and benzyl alcohol, and it was named benzoyl-CoA: benzyl alcohol O-benzoyltransferase (PtBEBT). Both enzymes were also capable of synthesizing plant volatile alcohol esters, such as hexenyl benzoate, at trace levels. Although the activities demonstrated are consistent with roles in salicinoid biosynthesis, direct tests of this hypothesis using transgenic poplar must still be performed.
authors.affiliation: Centre for Forest Biology, Department of Biology, University of Victoria, P.O. Box 3020, STN CSC, Victoria, BC V8W 3N5, Canada.
authors.forename: Russell J
authors.initials: RJ
authors.lastname: Chedgy
authors.affiliation: Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany.
authors.forename: Tobias G
authors.initials: TG
authors.lastname: Köllner
authors.affiliation: Centre for Forest Biology, Department of Biology, University of Victoria, P.O. Box 3020, STN CSC, Victoria, BC V8W 3N5, Canada. Electronic address: cpc@uvic.ca.
authors.forename: C Peter
authors.initials: CP
authors.lastname: Constabel
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D001565:Benzoates / Q000737:chemistry / Q000378:metabolism; D001592:Benzyl Alcohols / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005960:Glucosides / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism*; D015394:Molecular Structure; D010636:Phenols / Q000378:metabolism; D018515:Plant Leaves / Q000378:metabolism; D032107:Populus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000214:Acyl Coenzyme A; D001565:Benzoates; D001592:Benzyl Alcohols; D018076:DNA, Complementary; D005960:Glucosides; D006027:Glycosides; D010636:Phenols; C095421:tremulacin; C005696:salicin; C037817:benzoyl-coenzyme A; C022903:cinnamoyl-coenzyme A; D000217:Acyltransferases; C010631:salicyl alcohol; C006723:benzyl benzoate; C113068:salicortin
keywords: Benzoyltransferase; Escherichia coli heterologous expression; Herbivore defense; Poplar; Populus trichocarpa; Salicaceae; Salicinoid phenolic glycosides