Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549873
Title: Biotransformations with plant coenzyme A-dependent acyltransferases
Author: Cunningham, Oliver David
ISNI:       0000 0001 3400 4091
Awarding Body: Durham University
Current Institution: Durham University
Date of Award: 2008
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
The acylation of plant natural products is widely observed in nature and this modification often confers novel bioactivities. In nature, each of these acylations is selectively catalysed by a coenzyme A-dependent acyltransferase and biotransformations with these enzymes offer several potential advantages, including pre-disposed regio- and substrate- selectivity, reduced side-reactions and increased yield. The enzymatic introduction of non-natural acyl groups into natural product biosynthesis has the potential to diversify the application of these acylating enzymes, with several examples of unnaturally acylated natural products being reported to have novel bioactivity. To explore the potential for biosynthesising such acylated- derivatives in vivo, feeding studies with both natural and fluorinated phenylpropanoids were carried out in Arabidopsis thaliana and Petunia hybrida. In petunia, feeding with natural phenylpropanoids caused hyper-accumulation of acylated flavonoid products. This enhancement was particularly apparent when spraying the plants with the phenylpropanoyl methyl ester. Using the methyl esters, 4- hydroxycinnamic acid and its 4- fluorocinnamic acid analogue were apparently incorporated into quercetin 3- O- diglycoside biosynthesis in petunia. However, only the endogenous phenylpropanoids were able to be incorporated into acylated- anthocyanin biosynthesis in arabidopsis. In order to understand the factors that govern substrate selectivity in coenzyme A- dependent acyltransfer, the associated enzymic mechanisms were investigated in vitro. Plants utilise a coenzyme A acyl donor substrate formed through the action of an ATP-dependent CoA ligase, and the respective enzyme (At4CLl) from Arabidopsis was cloned and expressed in E. coli. The purified enzyme was shown to form a diverse range of CoA esters with phenylpropanoid derivatives bearing 4- or 3- 4- hydro, hydroxyl, fluoro and / or methoxy groups, in addition to 4- azidocinnamic acid substituents. These aromatic substituents were found to have a strong influence upon acyl substrate selectivity. A CoA-dependent acyltransferase which acylates cyanidin 3- 5- O- diglucoside in Gentiana triflora with phenylpropanoids was also cloned, expressed in E. coli and biochemically characterised. The acyltransferase was able to transfer various 4- and 3- 4- substituted phenylpropanoids. However, in this instance substrate selectivity was not as strongly influenced by phenylpropanoid aromatic substitution, which was indicative of a 'pre-screening' role of the CoA ligase with respect to acyl substrates. The in vitro biosynthesis of acylated flavonoids was optimised to transfer aromatic acids onto natural product acceptors using a one-pot biosynthetic approach, combining 4CL and acyltransferase activities. Consequently, coenzyme A was able to be efficiently re-cycled and was used on a much reduced scale. In addition, new strategies have been developed to isolate interesting acyltransferase biocatalysts based on their inhibition by a biotinylated fluorophosphonate probe. This approach showed that it was possible to identify CoA-dependent acyltransferases from crude protein preparations by recovering affinity labelled enzymes using a streptavidin sepharose solid support. The reactivity of the probe toward BAHD acyltransferases could be enhanced in the presence of UV irradiation, whilst labelling was abolished in the presence of the acyl acceptor. This afforded the ability to isolate enzyme activities according to their substrate recognition.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.549873  DOI: Not available
Share: