Inosine-5′-monophosphate dehydrogenase (IMPDH) is ubiquitously essential for the biosynthesis of guanosine nucleotides and catalyzes the NAD-dependent oxidation of IMP to XMP. IMPDH is a major target in the design of both antitumor and immunosuppresive drugs. The bacterial IMPDHs exhibit special properties compared to their eukaryotic counterparts but are poorly characterized.
IMPDH shares a two-domain organization composed of one catalytic domain, a (ß/a)8 barrel, and a smaller flanking domain, containing two CBS modules (forming together the so-called Bateman domain). Recent report revealed a moonlighting function in drosophila, where IMPDH was found to be a DNA-binding transcriptional repressor. Up to now, IMPDHs have been reported as tetrameric enzymes harbouring a catalytic domain and a tandem of cystathionine-ß-synthase (CBS) modules. The latter have no precise function assigned despite their nearly absolute conservation among IMPDHs and consistent evidence of their importance in vivo.
A multidisciplinary approach allowed us to demonstrate that the Pseudomonas aeruginosa IMPDH is octameric and allosterically regulated by MgATP via each CBS module (Labesse et al, 2013).
A. IMPDH functional organization (numbers correspond to the P. aeruginosa IMPDH): in blue, the catalytic domain and in green the Bateman domain containing two CBS modules. Our work shed light on the functional and structural role of this second domain, and has resulted in the proposal of a new paradigm for IMPDH mechanism of action. A variant (∆CBS) of the P. aeruginosa IMPDH, which Bateman domain has been deleted by linking alanine 92 directly to lysine 202, has been constructed by site-directed mutagenesis. B. Catalytic activity of the P. aeruginosa IMPDH as a function of IMP in the absence (red curve) and in the presence (blue curve) of 3 mM MgATP, indicating cooperative kinetics: this property had never been reported so far for any IMPDH. Two other bacterial IMPDHs exhibiting the same characteristics as the P. aeruginosa IMPDH ones have been identified. C. Catalytic activity of the ∆CBS variant as a function of IMP in the absence (red curve) of 3 mM MgATP. This variant exhibited the same catalytic properties as the R form, demonstrating the role of the Bateman domain into the allosteric regulation by MgATP. D. Structure of the P. aeruginosa IMPDH with its positive effector (purple spheres) showing the octamer (dimer of tetramers), and the effector binding sites within the Bateman domains (same code color as in A). The octameric organization, that has ben overlooked up to now, is drastically modified upon MgATP addition (as evidenced by cryo-EM experiments).
Revisiting deposited structural data we have found this newly discovered octameric organization conserved in other IMPDH structures (Labesse et al, 2013). Moreover we have shown that the human IMPDH1 formed two distinct octamers that can pile up into isolated fibres in the presence of MgATP while its pathogenic mutant D226N appeared to form massively aggregating fibres. The dramatic impact of this mutation could explain the severe retinopathy adRP10. Closely related to these first findings, the biochemical and physico-chemical characterization of seven other bacterial IMPDHs has been performed, among which six were totally unexplored (Alexandre et al, 2015). A classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures was established. Class I IMPDHs (including the P. aeruginosa IMPDH) are cooperative enzymes for IMP, which are activated by MgATP and are octameric in all tested conditions. On the other hand, class II IMPDHs behave as Michaelis-Menten enzymes for both substrates and are tetramers in their apo state or in the presence of IMP, which are shifted to octamers in the presence of NAD or MgATP. This work provides new insights into the IMPDH functional regulation and revealed for the first time that IMPDH has an octameric architecture modulated by MgATP binding to the CBS modules. Targeting the conserved effector binding pockets identified within the CBS modules might be promising to develop novel antibacterial compounds (see Inhibitors of Pseudomonas aeruginosa IMPDH project) or to counter the onset of IMPDH-related severe retinopathies.
