Evidence for p-hydroxybenzoate formation involving enzymatic phenylpropanoid side-chain cleavage in hairy roots of Daucus carota.
In this study, methyl jasmonate (MJ)-elicited hairy root cultures of Daucus carota were explored to study the enzymatic route to p-hydroxybenzoic acid (p-HBA) biosynthesis. Treatment with 100microM MJ caused an enhanced accumulation of p-HBA as well as total phenolic content in elicited root lines as compared to untreated (controls) lines. Using cell-free extract as the source of crude enzymes, attempt was made to reveal the enzymatic route to p-HBA formation. The accumulation of p-HBA was preceded by a substantial upliftment of p-hydroxybenzaldehyde dehydrogenase (HBD) activity in elicited lines as compared to controls. A rapid 6-fold enhancement of phenylalanine ammonia-lyase (PAL) activity, the first enzyme of the phenylpropanoid pathway was also observed. Finally, we demonstrated here for the first time, in D. carota, the evidence of a quite unusual p-hydroxybenzaldehyde synthase (HBS)-type enzyme, which catalyzes the penultimate step of p-HBA biosynthesis by making phenylpropanoid side-chain cleavage of p-coumaric acid without involvement of any cofactor(s), but uplifted by supplementation of a thiol reagent such as DTT in the reaction buffer. This enzyme showed activity in a relatively broad pH range (7-8.4) and the temperature optimum was found to be at 34 degrees C. The MJ-treated roots showed highest HBS activity at 24h (52nkat/mg protein), which was nearly 5-fold higher than that in the control lines.
Agricultural & Food Engineering Department, Natural Product Biotechnology Group, Indian Institute of Technology Kharagpur, Kharagpur, India.
Debabrata
D
Sircar
Adinpunya
A
Mitra
D000085:Acetates / Q000494:pharmacology; D000445:Aldehyde Oxidoreductases / Q000378:metabolism; D055162:Biocatalysis / Q000187:drug effects; D019755:Carbon-Carbon Lyases / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003373:Coumaric Acids / Q000737:chemistry / Q000378:metabolism; D003517:Cyclopentanes / Q000494:pharmacology; D018552:Daucus carota / Q000187:drug effects / Q000201:enzymology*; D054883:Oxylipins / Q000494:pharmacology; D010226:Parabens / Q000378:metabolism*; D010636:Phenols / Q000378:metabolism*; D010650:Phenylalanine Ammonia-Lyase / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000201:enzymology*; D011422:Propionates; D013997:Time Factors
D000085:Acetates; D003373:Coumaric Acids; D003517:Cyclopentanes; D054883:Oxylipins; D010226:Parabens; D010636:Phenols; D010936:Plant Extracts; D011422:Propionates; C072239:methyl jasmonate; D000445:Aldehyde Oxidoreductases; C068611:4-hydroxybenzaldehyde dehydrogenase; D019755:Carbon-Carbon Lyases; D010650:Phenylalanine Ammonia-Lyase; C495469:p-coumaric acid; C038193:4-hydroxybenzoic acid