Structural Characterisation of members of the DHDPS/NAL subfamily of proteins (2017)
Type of ContentTheses / Dissertations
Thesis DisciplineBiological Sciences
Degree NameMaster of Science
PublisherUniversity of Canterbury
AuthorsMacDonald, Cameronshow all
The DHDPS/NAL subfamily of enzymes share a very similar structure while catalysing a wide variety of reactions in different biochemical pathways. The structural similarity between members allows us to understand how this subfamily of enzymes has evolved. This project characterises some of the more obscure enzymes in the DHDPS/NAL subfamily in order to investigate evolutionary relationships within this subfamily of proteins. This will be done though techniques to investigate the structural states of these proteins as well as their solution based structure.
There are two protein groups under investigation: trans-hydroxybenzylidenepyruvate hydratase-aldolase (tHBP-HA), 1-pyrroline-4-hydroxy-2-carboxylate deaminase (HypD). tHBPHA catalyses the final step of the naphthalene degradation pathway, it has been previously characterised as a trimer. Analytical ultracentrifugation (AUC) has been used to probe the oligomeric states of the proteins to confirm if they are similar to the tetrameric DHDPS/NAL. tHBPHA was found to be consistent with a tetrameric protein. This was further shown through use of Small angle x-ray scattering (SAXS). Structure for tHBP-HA from P. fluorescens was solved through X-ray crystallography to 2.2Â, allowing for investigation of the active site. The replacement of a tyrosine from the adjacent monomer was the only difference in the conserved amino acids within the active site. It was found that tHBPHA does not show activity towards the DHDPS substrates.
The second protein investigated is HypD. HypD is involved in L-Hydroxyproline metabolism in bacteria and different groups have labelled it as either a tetrameric or hexameric protein. AUC showed that the four HypD proteins were consistent with hexameric proteins and were largely different to the reference protein E. coli DHDPS. This was again further shown using SAXS. The crystal structure for M. lupine HypD was produced to 2.9Å. The protein fits the hexameric architype, further showing that HypD are hexameric. The active site showed no interaction with the opposing monomer in the dimer interface, a contrast to the other members of the DHDPS/NAL subfamily. This shows that HypD is a novel member of the DHDPS/NAL subfamily. It was found that HypD does not show activity towards the DHDPS substrates.