Two classes of bacterial IMPDHs according to their quaternary structures and catalytic properties

Abstract

Inosine-5'-monophosphate dehydrogenase (IMPDH) occupies a key position in purine nucleotide metabolism. In this study, we have performed the biochemical and physico-chemical characterization of eight bacterial IMPDHs, among which six were totally unexplored. This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures. Class I IMPDHs 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. Our work provides new insights into the IMPDH functional regulation and a model for the quaternary structure modulation is proposed.

Fig 1. Primary sequence alignment of eight bacterial IMPDHs given by their acronym and explored in this work.

Identical residues are shaded. CBS modules of IMPDHpa are marked out by arrows. IMPDHpa residues involved in the interaction with ATP, Mg and IMP are marked out by red, green and yellow diamonds, respectively. The catalytic cysteine residue is marked out by a red diamond. ba: Bacillus anthracis; bt: Burkholderia thailandensis; kp: Klebsiella pneumonia; lpp: Legionella pneumophila subsp. pneumophila; nm: Neisseria meningitidis; pa: Pseudomonas aeruginosa; sa: Staphylococcus aureus.

Fig 2. Continuous sedimentation coefficient distribution analysis of IMPDHbt (A), IMPDHlpp (B) and IMPDHkp (C) at 1 mg/mL (corresponding to 18.5, 18.3 and 18.4 μM, respectively) in the absence (red curves) or presence of 3 mM MgATP (blue curves).

Sedimentation coefficients (S_{20, w}) are expressed in Svedberg units (1S = 10^-13s) and c(S) corresponds to normalized continuous size distribution.

Fig 3. IMPDHbt (A) and IMPDHlpp (B) activities versus NAD concentration.

Enzyme activity was determined at a fixed concentration of IMP (1 mM for IMPDHbt and 2 mM for IMPDHlpp), and in the absence (red curve) or in the presence (blue curve) of 5 mM MgATP. The curves correspond to the fit of the experimental data to the Michaelis-Menten equation and the calculated parameters are displayed in Table 4.

Fig 4. IMPDHbt (A) and IMPDHlpp (B) activity versus IMP concentration.

Enzyme activity was determined at a fixed concentration of NAD (2 mM for IMPDHbt and 6 mM for IMPDHlpp), and in the absence (red curve) or in the presence (blue curve) of 5 mM MgATP. The curves correspond to the fit of the experimental data to the Michaelis-Menten equation for IMPDHbt and Hill equation for IMPDHlpp and the calculated parameters are displayed in Table 5.

Fig 5. IMPDHbt (A), IMPDHba (B), IMPDHkp (C), IMPDHsa (D), IMPDHlpp (E) and IMPDHnm (F) activity versus MgATP concentration.

Reaction rates were determined at constant IMP and NAD concentrations (respectively, 0.05 mM and 0.3 mM for IMPDHbt, 0.5 mM and 2 mM for IMPDHba, 0.05 mM and 0.8 mM for IMPDHkp, 0.3 mM and 1.5 mM for IMPDHsa, 0.5 mM and 0.5 mM for IMPDHlpp and IMPDHnm).

Fig 6. ANS binding to IMPDHpa and IMPDHbt.

(A) Left panel: ANS fluorescence spectra alone at 10 μM (green), in the presence of 1 μM IMPDHpa without (red) or with 100 μM ATP (blue). Right panel: fluorescence intensity measured at 495 nm as a function of MgATP concentration. (B) The same curves for IMPDHbt.

Fig 7. Proposed model of the modulation of the quaternary structure of class I and class II IMPDHs in the absence (APO) or in the presence of MgATP.

For each tetramer, the core domain is represented as a cone and the CBS modules are on the side. MgATP are shown as red circles. Class I IMPDHs adopt an octameric architecture where the CBS modules of two tetramers interplay together in. MgATP induces a global shape rearrangement without disturbing the degree of oligomerization. Class II IMPDHs are tetrameric in the apo state. Different conformations of the tetramers would coexist and the addition of MgATP would trigger the equilibrium towards tetrameric species, which would dimerize into octamers.