title: Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+-dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift.
abstract: A novel hydroxylase activity catalyzing the formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA was identified in crude extracts from cultured parsley cells. The extracts were less active (Vmax/Km) in converting trans-4-coumaric to trans-caffeic acid. Optimal hydroxylase activity was found at pH 6.5 with a steep decline toward both pH 7.4 and pH 5.0. The enzyme activity requires ascorbate and Zn2+ at optimal concentrations of 50 and 0.5 mM, respectively. No other reductant could replace ascorbate, whereas high concentrations of Ca2+ partially substituted for Zn2+. The enzyme is soluble and appears to be located in the cytoplasm. The unusual pH optimum suggests that the hydroxylase is inactive at the normal cytoplasmic pH. Upon treatment of parsley cells with an elicitor derived from Phytophthora megasperma f. sp. glycinea, the cytoplasmic pH dropped by approximately 0.25 pH unit within 55 min as determined by 31P NMR spectroscopy. Our results suggest that this shift in the cytoplasmic pH is sufficient for the activation of the hydroxylase, eventually leading to the formation of caffeoyl and feruloyl esters. Such esters may be a part of a very rapid resistance response of the plant cells, which would leave no time for de novo enzyme synthesis.
authors.affiliation: Biologisches Institut II der Universität Freiburg, Biochemie der Pflanzen, Federal Republic of Germany.
authors.forename: R E
authors.initials: RE
authors.lastname: Kneusel
authors.forename: U
authors.initials: U
authors.lastname: Matern
authors.forename: K
authors.initials: K
authors.lastname: Nicolay
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism*; D002478:Cells, Cultured; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D015032:Zinc / Q000494:pharmacology
chemical_list: D000214:Acyl Coenzyme A; C058644:4-coumaroyl-coenzyme A; C058645:caffeoyl-coenzyme A; D006899:Mixed Function Oxygenases; C058643:4-coumaroyl-coenzyme A 3-hydroxylase; D015032:Zinc