Inosine monophosphate dehydrogenase (IMPDH) catalyses the key step in de novo guanine nucleotide biosynthesis at the branch point of GTP and ATP production. Mammals have two ubiquitous, catalytically indistinguishable isoforms, IMPDH type I and type II, and these are considered functionally interchangeable. Each contains a Bateman domain known to serve as energy-sensing / allosteric regulatory modules in a range of unrelated proteins. Mutations in the Bateman domain of type I, which do not affect catalytic activity, cause the retina-degenerative disease, retinitis pigmentosa (RP). The central hypothesis of this thesis is that IMPDH is regulated. In particular, that regulation occurs in an isoform specific manner and that mutations causal to RP affect enzyme regulation. Here we have visualised, including in real-time, the redistribution or clustering of IMPDH into linear macrostructures in a time-dependent manner which appeared to be intimately associated with changes in intracellular nucleotide levels. Data presented suggest the significance of IMPDH clustering is unlikely to be associated with substrate channelling, via interaction with other proteins in the de novo biosynthesis pathway, or enhanced protein stability. Although both isoforms responded similarly to fluctuations in intracellular nucleotide levels, type I had a higher propensity to spontaneously cluster into macrostructures compared to type II. This propensity to cluster was found to be conferred by the N-terminal 244 amino acids, which includes the Bateman domain, using a series of type I / type II chimera proteins. A comparative and novel approach revealed isoform-specific purine nucleotide binding characteristics. Type I bound ATP and type II bound AMP, via a mechanism involving the Bateman domain, resulting in conformational changes in IMPDH. This nucleotide binding was not associated with allosteric activation of IMPDH catalytic activity. The RP-causing mutation, R224P, abolished ATP binding and this correlated with an altered propensity to cluster. Collectively these data (i) show IMPDH distribution is regulated by the intracellular environment (ii) demonstrate that the IMPDH isoforms are modulated in a differential manner by AMP and ATP by a mechanism involving the Bateman domain, (iii) indicate communication between the Bateman domain and the active site and (iv) demonstrate that a RP-causing mutation compromises such regulation. From a broader perspective, this work raises the possibility that the nucleotide sensing properties of the Bateman domain in IMPDH serve to regulate IMPDH and co-ordinate nucleotide homeostasis, thereby giving rise to cellular plasticity in an isoform-specific manner to meet the requirements of the cellular environment.
| Identifer | oai:union.ndltd.org:ADTP/284404 |
| Creators | Elaine Thomas |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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