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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

Functional Analysis of the Amine Substrate Specificity Domain of Pepper Tyramine and Serotonin N-Hydroxycinnamoyltransferases¹

Department of Molecular Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Gwangju 500–757, Korea (S.K., K.K., G.C.C., K.B.); Korea Research Institute of Bioscience and Biotechnology, Daejeon 305–600, Korea (D.C.); Division of Applied Life Sciences, Graduate School of Kyoto University, Kyoto 606–8502, Japan (A.I.); and Structural Biology Center, Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030 (D.-S.L.)

Pepper (Capsicum annuum) serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N-hydroxycinnamic acid amides of serotonin, including feruloylserotonin and p-coumaroylserotonin. To elucidate the domain or the key amino acid that determines the amine substrate specificity, we isolated a tyramine N-hydroxycinnamoyltransferase (THT) gene from pepper. Purified recombinant THT protein catalyzed the synthesis of N-hydroxycinnamic acid amides of tyramine, including feruloyltyramine and p-coumaroyltyramine, but did not accept serotonin as a substrate. Both the SHT and THT mRNAs were found to be expressed constitutively in all pepper organs. Pepper SHT and THT, which have primary sequences that are 78% identical, were used as models to investigate the structural determinants responsible for their distinct substrate specificities and other enzymatic properties. A series of chimeric genes was constructed by reciprocal exchange of DNA segments between the SHT and THT cDNAs. Functional characterization of the recombinant chimeric proteins revealed that the amino acid residues 129 to 165 of SHT and the corresponding residues 125 to 160 in THT are critical structural determinants for amine substrate specificity. Several amino acids are strongly implicated in the determination of amine substrate specificity, in which glycine-158 is involved in catalysis and amine substrate binding and tyrosine-149 plays a pivotal role in controlling amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of tyrosine is corroborated by the THT-F145Y mutant that uses serotonin as the acyl acceptor. The results from the chimeras and the kinetic measurements will direct the creation of additional novel N-hydroxycinnamoyltransferases from the various N-hydroxycinnamoyltransferases found in nature.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kyoungwhan Back (kback{at}chonnam.ac.kr).

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.071514.

^* Corresponding author; e-mail kback{at}chonnam.ac.kr; fax 82–62–530–2169.

Received September 13, 2005; returned for revision November 14, 2005; accepted November 28, 2005.