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Title: An exploration of phosphorylases for the synthesis of carbohydrate polymers
Author: O'Neill, Ellis
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2013
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Phosphorylases are interesting enzymes with regard to both their role in metabolism and their use in the in vitro synthesis of carbohydrates. The disaccharide phosphorylases have attracted attention because of their strict stereo- and regiospecificity and their tractability. The polymerising phosphorylases have received less attention due to heterogeneous product formation, requiring more complex analyses. In this work three polymerising carbohydrate phosphorylases have been studied. The plant α-1,4-glucan phosphorylase PHS2 is closely related to the well characterised mammalian glycogen phosphorylase. We present the first crystal structures of the plant enzyme which reveals a unique surface binding site. PHS2 allowed the production of novel starch like surface, both in two and three dimensions, which show some of the same properties as a native starch granule. This can now be used to study starch-active enzymes on an insoluble glucan surface which is analogous to the native starch granule. The bacterial β-1,4-glucan phosphorylase CDP is involved in degradation of cellulose. In the reverse direction this enzyme allows the rapid synthesis of cellulose polymers in solution and also allows the synthesis of hemicellulose-like materials. The substrate specificity can in part be probed in the crystal structure presented here, which represents the first structure of a polymerising, inverting phosphorylase. Together these data provide the foundation for further work with this enzyme in the synthesis of plant cell wall related glycans. The third enzyme studied was the β-1,3-glucan from the unsequenced alga Euglena gracilis, which was used for the facile enzymatic synthesis of β-glucosyl glycerols. In order to identify the sequence of this enzyme we obtained de novo transcriptome sequencing data from this alga, which has revealed unexpected metabolic diversity. Aside from complex carbohydrate metabolism, there are also many surprising features, including novel enzyme architectures, antioxidants only previously noted in human parasites and complex natural product synthases.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available