Modification of biologically important natural product scaffolds through biocatalysis, using Taxus acyltransferases

The antineoplastic agents paclitaxel (TaxolRTM) and docetaxel (TaxotereRTM) are currently supplied commercially by plant cell fermentations which rely on a biological source. The production of paclitaxel and related compounds can therefore be improved by understanding the biosynthesis of these metabolites in detail. Structure-activity-relationship studies have led to the development of highly promising paclitaxel analogues compared to the parent molecule through acyl group modifications, which are currently obtained by in-effective semisynthetic methods. Instead, catalysis of Taxus acyltransferases belonging to the BAHD plant superfamily can potentially be applied to developing the biotechnological production of paclitaxel and its analogues. Thus, the potential biocatalytic applications of a 2-O-benzoyltransferase (TBT) and a 13-O-3-amino-3-phenylpropanoyltransferase (BAPT) involved in paclitaxel biosynthesis were studied.;Two site-directed mutations within the wild-type 2-O-benzoyltransferase cDNA (tbt), from Taxus cuspidata plants, yielded an encoded protein containing replacement amino acids at Q19P and N23K that map to a solvent-exposed loop region. The likely significant changes in the biophysical properties invoked by these mutations caused the overexpressed, modified TBT (mTBT) to partition into the soluble enzyme fraction about 5-fold greater than the wild-type enzyme. Sufficient protein could now be acquired to examine the scope of the substrate specificity of mTBT by incubation with 7,13-O,O-diacetyl-2- O-debenzoylbaccatin III that was mixed individually with various substituted benzoyls, alkanoyls, and (E)-butenoyl CoA donors. The mTBT catalyzed production of several 7,13-O,O -diacetyl-2-O-acyl-2-O-debenzoylbaccatin III analogues demonstrated the broad specificity of mTBT, suggesting that a plethora of 2-O-acyl variants of the antimitotic paclitaxel can be assembled through biocatalytic sequences.;The scope of the taxane co-substrate specificity of mTBT hydrolysis was examined by incubating CoASH with various taxane co-substrates. The mTBT hydrolysis was highly regiospecific to the C-2 benzoyl moiety. However, the substrate scope was fairly limited. The 2-O-benzoylated taxoids hydroxylated at C-7 and/or both C7 and C13 were not productive in mTBT hydrolytic reaction, suggesting that the hydroxylation at C-7 precludes the mTBT hydrolysis. This observation was verified by examining the taxane co-substrate specificity of mTBT acylation and further analysis allowed for pinpointing the relative timing of 2-O-benzoylation and the 7beta-hydroxylation steps in the overall paclitaxel pathway.;The wild-type 13-O-3-amino-3-phenylpropanoyltransferase cDNA (bapt), from Taxus cuspidata plants was recombinantly expressed to produce BAPT. Several arylpropanoyl CoA thioesters were incubated with baccatin III and purified BAPT to examine the substrate specificity; each formed the corresponding 13-O-arylpropanoylbaccatin III analogue. Also, the mechanistic studies of BAPT demonstrated the substrate-assisted catalysis of BAPT.;The conducted substrate specificity studies of Taxus acyltransferases suggest the potential application of Taxus acyltransferases in developing biotechnological production of paclitaxel and its analogues. The fine details of paclitaxel biosynthesis are also advantageous in improving the production of paclitaxel through plant cell fermentations. Moreover, a variety of directed evolutionary analyses can be employed to potentially produce new catalyst derivatives that are able to transfer an even greater or more refined scope of novel acyl groups to the taxane core or other diterpene scaffolds.