Characterisation of Enzyme Evolution through Ancestral Enzyme Reconstruction

Prentice, Erica Jean

Characterisation of Enzyme Evolution through Ancestral Enzyme Reconstruction

dc.contributor.advisor	Arcus, Vickery L.
dc.contributor.author	Prentice, Erica Jean
dc.date.accessioned	2014-08-01T03:25:40Z
dc.date.available	2014-08-01T03:25:40Z
dc.date.issued	2013
dc.date.updated	2013-10-24T22:04:31Z
dc.description.abstract	Through ancestral sequence reconstruction (ASR) techniques, ancient enzymes can be recreated and biochemically tested, giving insight into the enzymes’ evolutionary history. A previous study by Hobbs et al. (2012) has shown that some ancestral 3-isopropylmalate dehydrogenase (IPMDH) enzymes of the Bacillus lineage are more catalytically efficient and kinetically stable than extant counterparts. Given these characteristics, this trend raises questions as to why ancestral Bacillus IPMDH enzymes have been superseded by catalytically slower and less kinetically stable counterparts. The homology between IPMDH and the dehydrogenases of tartrate, malate and isocitrate makes IPMDH an interesting model enzyme in terms of the evolution of substrate specificity. Here, the reconstruction of a 2.7 billion year old enzyme has been attempted to extend the reconstruction of IPMDH back to the last common ancestor of the Firmicutes. This reconstruction tested the limits of ASR techniques in terms of time and levels of sequence divergence, especially for such a structurally complex enzyme. However, upon expression and purification, the enzyme was found to form an inactive, soluble aggregate. This suggests that current ASR techniques are too simplistic to reconstruct the complexity and divergence of IPMDH back as far as the last common ancestor of the Firmicutes. Enzyme evolution was investigated with ancestors from the Bacillus genus. Substrate promiscuity of ancestral enzymes was compared to a contemporary counterpart. It was concluded that the ancestral IPMDH enzymes tested do not show additional substrate promiscuity when compared to contemporary counterparts. The fitness of organisms carrying the IPMDH ancestors was assessed to establish what effects the high turnover rates and kinetic stability possessed by some ancestral IPMDH enzymes had on cells when functioning within the normal catalytic pathway for leucine. In vivo, the fastest and most kinetically stable ancestral IPMDH resulted in slower growth rates. This detrimental effect in vivo clarifies why this enzyme has been lost over evolutionary time. The X-ray crystal structure of the most recent IPMDH ancestor was also determined at 2.6 Å resolution. The structure of this ancestral IPMDH was found to be similar to other IPMDH structures, including the previously solved IPMDH from the last common ancestor of the Bacillus.
dc.format.mimetype	application/pdf
dc.format.mimetype	application/vnd.openxmlformats-officedocument.spreadsheetml.sheet
dc.identifier.citation	Prentice, E. J. (2013). Characterisation of Enzyme Evolution through Ancestral Enzyme Reconstruction (Thesis, Master of Science (MSc)). University of Waikato, Hamilton, New Zealand. Retrieved from https://hdl.handle.net/10289/8717	en
dc.identifier.uri	https://hdl.handle.net/10289/8717
dc.language.iso	en
dc.publisher	University of Waikato
dc.rights	All items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subject	ancestral sequence reconstructio
dc.subject	isopropylmalate dehydrogenase
dc.title	Characterisation of Enzyme Evolution through Ancestral Enzyme Reconstruction
dc.type	Thesis
pubs.place-of-publication	Hamilton, New Zealand	en_NZ
thesis.degree.grantor	University of Waikato
thesis.degree.level	Masters
thesis.degree.name	Master of Science (MSc)