title: Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia).
abstract: Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.
authors.affiliation: Department of Plant Sciences, University of California, Davis, CA 95616-8683, USA.
authors.forename: Ryann M
authors.initials: RM
authors.lastname: Muir
authors.forename: Ana M
authors.initials: AM
authors.lastname: Ibáñez
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Uratsu
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Ingham
authors.forename: Charles A
authors.initials: CA
authors.lastname: Leslie
authors.forename: Gale H
authors.initials: GH
authors.lastname: McGranahan
authors.forename: Neelu
authors.initials: N
authors.lastname: Batra
authors.forename: Sham
authors.initials: S
authors.lastname: Goyal
authors.forename: Jorly
authors.initials: J
authors.lastname: Joseph
authors.forename: Eluvathingal D
authors.initials: ED
authors.lastname: Jemmis
authors.forename: Abhaya M
authors.initials: AM
authors.lastname: Dandekar
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism; D056148:Chromatography, Reverse-Phase; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005707:Gallic Acid / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D031324:Juglans / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D012765:Shikimic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
chemical_list: C096387:3-dehydroshikimate; D012765:Shikimic Acid; D005707:Gallic Acid; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase