Purification, characterization and partial amino acid sequencing of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase from tobacco cell-suspension cultures.
We report the purification of hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase (THT) to apparent homogeneity in 12% yield from tobacco (Nicotiana tabacum L. cv. Xanthi) cell-suspension cultures elicited with a commercial preparation of pronase. The purification procedure employs only four chromatography steps and takes advantage of the fact that the transferase binds tightly both to phenyl-Sepharose and to hydroxyapatite. The native enzyme has a pI of 5.2 and consists of two identical or very similar subunits of approximately 24 kDa. The purified enzyme can synthesise a wide range of amides due to its relatively low specificity for cinnamoyl-CoA derivatives and hydroxyphenethylamines, but its best substrates are tyramine and feruloyl-CoA. THT follows Michaelis-Menten kinetics in the presence of low concentrations of feruloyl-CoA but negative cooperativity occurs when this concentration increases above 2.5 microM, resulting in a marked decrease of the affinity for tyramine. Large deviations from Michaelis-Menten kinetics are also observed when 3-methoxytyramine is used as acyl acceptor. The activity of tobacco THT is not affected by the addition of CaCl2 or MgCl2 but its maximal velocity is increased up to twofold by addition of ethanol to the assay mixture. It is inhibited in vitro by L-tyrosine benzyl ester, which binds reversibly to the tyramine-binding site. Experiments performed using L-tyrosine benzyl ester and caffeoyl-CoA as inhibitors confirm that feruloyl-CoA is the first substrate to add to the transferase in an ordered bi-bi mechanism. Part of the amino acid sequence of the transferase, elucidated by microsequencing of tryptic peptides, is also described.
Negrel
J
J
Laboratoire de Phytoparasitologie, INRA-CNRS, INRA BV 1540, Dijon, France.
Javelle
F
F
D000217:Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010947:Plants, Toxic; D013379:Substrate Specificity; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology*
D000217:Acyltransferases; C108168:hydroxycinnamoyl-CoA-tyramine N-(hydroxycinnamoyl)transferase