Different Characteristics and Nucleotide Binding Properties of Inosine Monophosphate Dehydrogenase (IMPDH) Isoforms

Robert G. Parton,Anthony George,Jennifer H. Gunter,Julie A. Webster,Elaine C. Thomas,Nicole L. Schieber,Viola Oorschot,Jonathan P. Whitehead

doi:10.1371/journal.pone.0051096

Abstract

We recently reported that Inosine Monophosphate Dehydrogenase (IMPDH), a rate-limiting enzyme in de novo guanine nucleotide biosynthesis, clustered into macrostructures in response to decreased nucleotide levels and that there were differences between the IMPDH isoforms, IMPDH1 and IMPDH2. We hypothesised that the Bateman domains, which are present in both isoforms and serve as energy-sensing/allosteric modules in unrelated proteins, would contribute to isoform-specific differences and that mutations situated in and around this domain in IMPDH1 which give rise to retinitis pigmentosa (RP) would compromise regulation. We employed immuno-electron microscopy to investigate the ultrastructure of IMPDH macrostructures and live-cell imaging to follow clustering of an IMPDH2-GFP chimera in real-time. Using a series of IMPDH1/IMPDH2 chimera we demonstrated that the propensity to cluster was conferred by the N-terminal 244 amino acids, which includes the Bateman domain. A protease protection assay suggested isoform-specific purine nucleotide binding characteristics, with ATP protecting IMPDH1 and AMP protecting IMPDH2, via a mechanism involving conformational changes upon nucleotide binding to the Bateman domain without affecting IMPDH catalytic activity. ATP binding to IMPDH1 was confirmed in a nucleotide binding assay. The RP-causing mutation, R224P, abolished ATP binding and nucleotide protection and this correlated with an altered propensity to cluster. Collectively these data demonstrate that (i) the isoforms are differentially regulated by AMP and ATP by a mechanism involving the Bateman domain, (ii) communication occurs between the Bateman and catalytic domains and (iii) the RP-causing mutations compromise such regulation. These findings support the idea that the IMPDH isoforms are subject to distinct regulation and that regulatory defects contribute to human disease.

Highlights

Inosine monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in the de novo biosynthesis of guanine nucleotides, which are essential for various cellular processes
Characterisation of Inosine Monophosphate Dehydrogenase (IMPDH) Macrostructures We previously demonstrated that redistribution of IMPDH to macrostructures may be promoted upon intracellular nucleotide depletion with mycophenolic acid (MPA), an IMPDH inhibitor, and more importantly with decoyinine, a specific inhibitor of the enzyme GMP synthetase which catalyses the reaction immediately downstream of IMPDH [9]
The three-dimensional image shows that the circular/annular macrostructures are true ‘‘donuts’’ and do not represent solid spheres with a core which is inaccessible to antibody. The ultrastructure of these macrostructures was further investigated by immuno-electron microscopy (EM) of ultra-thin cryosections of Chinese Hamster Ovary (CHO) cells treated with vehicle or MPA (Fig. 2)

Summary

Introduction

Inosine monophosphate dehydrogenase (IMPDH) catalyses the rate-limiting step in the de novo biosynthesis of guanine nucleotides, which are essential for various cellular processes. The proteins share 84% amino acid identity and virtually indistinguishable catalytic activity, as determined in vitro, but differ in their tissue expression [1,2,3,4]. Mutations in the IMPDH1 gene, but not IMPDH2, give rise to a number of closely related retinal diseases [7,8]. We observed striking differences in the propensity of IMPDH1 and IMPDH2 to cluster into filamentous spicules (1–2 mm) and ‘macrostructures’ (2– 10 mm), the two isoforms responded to gross changes in intracellular nucleotide levels [9]. We hypothesised that divergence in the tandem cystathionine b-synthase (CBS) domain-containing Bateman domain underpinned these differences and that disease-causing mutations situated in or around the Bateman domain compromised the regulation of IMPDH1 rather than its activity per se [10,11,12]

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