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Published on December 29, 2005; 10.1104/pp.105.071514

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Received September 13, 2005
Returned for revision October 31, 2005
Accepted November 28, 2005

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

Sei Kang , Kiyoon Kang , Gap Chae Chung , Doil Choi , Atsushi Ishihara , Dong-Sun Lee , and Kyoungwhan Back *

Department of Molecular Biotechnology, Agricultural Plant Stress Research Center, Biotechnology Research Institute, Chonnam National University, Gwangju, 500-757, Korea
Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, Korea
Division of Applied Life Sciences, Graduate School of Kyoto University, Kyoto 606-8502, Japan
Structural Biology Center, Department of Biochemistry and Molecular Biology, University of Texas Medical School, TX 77030, USA

* Corresponding author; email: kback{at}chonnam.ac.kr.

Pepper serotonin N-hydroxycinnamoyltransferase (SHT) catalyzes the synthesis of N- hydroxycinnamic acid amides (HCAAs) 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 Capsicum annuum. Purified recombinant THT protein catalyzed the synthesis of HCAAs 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 129-165 of SHT and the corresponding residues in THT, 125-160, are critical structural determinants for the amine substrate specificity. Several amino acids are strongly implicated in the determination of the amine substrate specificity, in which Gly-158 is involved in catalysis and amine substrate binding, and Tyr-149 plays a pivotal role in controlling the amine substrate specificity between serotonin and tyramine in SHT. Furthermore, the indisputable role of Tyr is corroborated by the THT-F145Y mutant that serotonin is accepted 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.






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