Untitled

Abstract

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1-3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt 1 but differing in the 3'-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyl-transferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.

The final acylation step in Taxol biosynthesis: Cloning of the taxoid C13-side-chain N -benzoyltransferase from Taxus

“…Biosynthetic analysis using Taxus brevifolia mature bark tissue as enzyme source demonstrated that N-benzoylation of 3Ј-debenzoyltaxol is the final step in the Taxol pathway (4). The substantial deduced sequence identity (58-69%) within a family of nine cloned acyltransferases from Taxus (10) [including a taxoid 2-O-benzoyltransferase among the three which have been functionally defined (17)] and between these and a Dianthus anthranilate N-hydroxycinnamoyl͞benzoyltrans-ferase (Ϸ50-57%) (18) suggested that one of the six remaining undefined Taxus clones might encode the N-debenzoyltaxol N-benzoyltransferase.…”

Purification and characterization of benzoate:coenzyme A ligase from Clarkia breweri

“…C. breweri BZL catalyzes the thioesterification of benzoic acid to its corresponding CoA derivative. There are multiple examples in plant secondary metabolism that feature benzoic acid as a biosynthetic building block [1][2][3][4]. Benzoylation of these compounds is catalyzed by acyltransferases that require benzoyl-CoA as a substrate.…”

“…Benzoic acid is a component of many secondary metabolites in plants, including cocaine [1], taxol [2], dianthramide B [3], and some glucosinolates [4]. In these and other molecules, the benzoyl moiety is transferred by acyltransferases, requiring activated benzoyl-CoA as a substrate.…”

Molecular Characterization of the Salutaridinol 7-O-Acetyltransferase Involved in Morphine Biosynthesis in Opium Poppy Papaver somniferum

“…The radioactivity present on the TLC plates was localized and quantitated with a Rita Star TLC scanner (Raytest). The identity of the enzymic reaction product as thebaine was ascertained by HPLC-MS using a Finnigan MAT TSQ 7000 (electrospray voltage, 4.5 kV; capillary temperature, 220°C; carrier gas, N 2 ) coupled to a Micro-Tech Ultra-Plus Micro-LC equipped with an Ultrasep RP18 column ( (22); BEAT, benzylalcohol acetyltransferase from C. breweri (26), HCBT, anthranilate N-hydroxycinnamoyl/benzoyltransferase from D. caryophyllus (27); DBAT, 10-deacetylbaccatin III-10-O-acetyltransferase and TAT, taxadienol acetyltransferase, both from T. cuspidata (28,29). Black boxes indicate conserved residues; white boxes indicate the internal peptide sequences obtained from native salutaridinol 7-O-acetyltransferase; arrows indicate the positions of the peptides used to design oligodeoxynucleotide primers for RT-PCR; # denotes positions of the highly conserved consensus sequence HXXXD.…”

“…E, HPLC-positive ion electrospray mass spectral analysis of thebaine standard. alcohol acetyltransferase from Clarkia breweri (34%) (26), anthranilate N-hydroxycinnamoyl/benzoyltransferase from Dianthus caryophyllus (25%) (27), taxadienol acetyltransferase (24%), and 10-deacetylbaccatin III-10-O-acetyltransferase (22%) both from Taxus cuspidata (28,29).…”