Identification and characterization of cDNA clones encoding hydroxycinnamoyl‐CoA:tyramine N‐hydroxycinnamoyltransferase from tobacco

Abstract

The sequences of three cDNA clones that include the complete coding region of hydroxycinnamoylCoA:tyramine N-hydroxycinnamoyltransferase (THT) from tobacco are reported. The three cDNAs were isolated by antibody screening of a cDNA expression library produced from poly(A) + RNA purified from tobacco leaves (Nicotiana tabacum cv. Bottom Special), previously infiltrated with an incompatible strain of Ralstonia solanacearum. The identity of these clones was confirmed by the detection of THT activity in extracts of transformed Escherichia coli and by matching the translated polypeptides with tryptic enzyme sequences. cDNA clones tht4 and tht11 differ only by their 5 H leader and 3 H UTRs and therefore encode the same protein, whereas tht10 and tht11 exhibit 95 and 99% sequence identity at the DNA and deduced amino acid levels, respectively. The three clones encode proteins of 226 amino acids with calculated molecular masses of 26 kDa. The deduced amino acid sequences show no similarity with the sequence of anthranilate hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus, the only enzyme exhibiting hydroxycinnamoyltransferase activity to be cloned so far in plants. In contrast, comparison of the THT amino acid sequence with protein sequence databases revealed substantial homology with mammalian diamine acetyltransferases. The THT clones hybridized to a 0.95-kb mRNA from elicited tobacco cell-suspension cultures and also to a mRNA of similar size from wound-healing potato tubers. The messengers for THT were also found to be expressed at relatively high levels in tobacco root tissues. Southern hybridization of tobacco genomic DNA with THT cDNA suggests that several copies of the THT gene occur in the tobacco genome. Inhibition experiments using amino-acid-specific reagents demonstrated that both histidyl and cysteyl residues are required for THT activity. In the course of these experiments THT was also found to be inhibited by (2-hydroxyphenyl) amino sulfinyl acetic acid 1,1-dimethylethyl ester, an irreversible inhibitor of cinnamyl alcohol dehydrogenase.

Identification of an Arabidopsis Feruloyl-Coenzyme A Transferase Required for Suberin Synthesis

“…Tyramine is sometimes listed as an aromatic component of suberin, being found as amide with phenylpropanoid acids (Kolattukudy, 2001;Bernards, 2002). However, genes for hydroxycinnamoyl-CoA:tyramine N-hydroxycinnamoyl transferases have been cloned and the proteins are members of the diamine acetyltransferase family (Farmer et al, 1999;Schmidt et al, 1999), so there is no need to postulate ASFT as an N-acyltransferase. Our Arabidopsis seed polyester monomer analyses were set up to recover tyramine, but none was observed (Molina et al, 2006).…”

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

“…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).…”

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

“…It has been well documented that THT enzymes have a broad range of substrate specificities and are encoded by a small gene family (Farmer et al, 1999;Schmidt et al, 1999). Tobacco and potato contain at least three THT isogenes, as shown by genomic Southern analyses.…”

“…AF329463) revealed 78% identity between the proteins. The protein has a high level of homology with THTs from other plants, including 83%, 80%, and 71% amino acid identity with tomato, potato, and tobacco THT proteins, respectively (Farmer et al, 1999;Schmidt et al, 1999;Von Roepenack-Lahaye et al, 2003). In the phylogenetic tree based on amino acid sequence of N-hydroxycinnamoyltransferases from four plant species, pepper SHT showed a greater evolutionary distance from pepper THT than other THTs within species (Fig.…”

“…Numerous THT enzymes from plant sources have been partially or completely purified and characterized (Farmer et al, 1999;Schmidt et al, 1999;Yu and Facchini, 1999;Ishihara et al, 2000). These enzymes behave as soluble proteins with a molecular mass of 26 to 28 kD.…”