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

Abstract

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.

Evolutionarily Distinct BAHD N-acyltransferases are Responsible for Natural Variation of Aromatic Amine Conjugates in Rice

“…While BAHD proteins are responsible for the acylation of aliphatic and arylmonoamine acceptors, GNAT enzymes are active on arylamines (Bassard et al, 2010). Although they are from two distinct protein families that show low sequence identity, the BAHD N-acyltransferases, Os-THTs and Os-TBTs, identified in our study demonstrate that proteins from both families can act on aryldiamines (Tables 2 and 3; Jang et al, 2004;Kang et al, 2006). Comparing the specificity of the enzymes between the two families showed that whereas GNAT members are highly specific for tyramine, the BAHD enzymes may be regarded as bifunctional tryptamine/tyramine N-acyltransferases.…”

“…Here, comparative sequence analysis with in vitro assays using mutant enzymes led to the identification of a RRRR motif that is sufficient to transform agmatine utilization ( Figures 9A and 9B), which is similar to the conserved arginine-rich motifs in RNA binding proteins (Lazinski et al, 1989;Zapp et al, 1991;Yuryev et al, 1996). This integrated approach has also been applied to identify key residues of enzymes involved in coumaroyl serotonin, terpene, and acylsucrose biosynthesis (Kang et al, 2006;Kang et al, 2014;Fan et al, 2016) and may be regarded as a general strategy in the structurefunction study of enzymes.…”

“…One of the first genes encoding this type of enzyme to be characterized was the gene encoding tyramine:N-hydroxycinnamoyl transferase (THT; Hohlfeld et al, 1995;Schmidt et al, 1999;Yu and Facchini, 1999;Back et al, 2001;Von Roepenack-Lahaye et al, 2003;Kang et al, 2006). Analysis of their amino acid sequences revealed that all these enzymes belong to the GCN5-related N-acetyltransferases (GNAT) protein family, which also includes mammalian spermidine/spermine acetyltransferase and several microbial antibiotic N-acetyltransferases (Lu et al, 1996;Farmer et al, 1999;Kang et al, 2006). In contrast to the situation where PAs of arylamine moieties are mainly biosynthesized by the GNAT family of enzymes, the biosynthesis of the aliphatic type of phenolamides is exclusively the domain of BAHD acyltransferases (D'Auria, 2006;Bassard et al, 2010).…”

Synthesis and Biological Evaluation of Piperic Acid Amides as Free Radical Scavengers and α-Glucosidase Inhibitors

“…Satisfactory yields were obtained in all cases. Piperic acid (2) was prepared by alkaline hydrolysis of piperine (1), by means of a previous procedure, 20) with modifications. Piperic acid amides (29 and 30) with aminoalkyl groups were synthesized by the condensation of the acid chloride (3) with the corresponding mono-Boc-diamines (26 and 28) followed by a deprotection step.…”

“…As an example, the biosynthesis of tyramine and serotonin derivatives are catalyzed by the action of tyramine-and serotonin-N-hyroxycinnamoyltransferase, respectively. 1) Recent studies have reported the tyrosinase inhibitory effects of synthetic hydroxycinnamic acid amides, derived from coupling of caffeic acid and its derivatives with biogenic amines, such as tyramine, dopamine, and serotonin. [2][3][4][5] In addition, serotonin derivatives have a number of biological effects, such as α-glucosidase inhibition, 6) antioxidative activity, 7) and cyclooxygenase-2 (COX-2) inhibition.…”