An exploration of phosphorylases for the synthesis of carbohydrate polymers

O'Neill, Ellis (2013) An exploration of phosphorylases for the synthesis of carbohydrate polymers. Doctoral thesis, University of East Anglia.

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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

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 5605 not found.
Date Deposited: 03 Feb 2014 15:11
Last Modified: 03 Feb 2014 15:11

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