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

Alexandre T, Raynal B, Rayna B, Munier-Lehmann H - PLoS ONE (2015)

Bottom Line: This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Chimie et Biocatalyse, Département de Biologie Structurale et Chimie, 28 rue du Dr Roux, F-75015, Paris, France; Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 3523, F-75015, Paris, France; Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.

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.

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Related in: MedlinePlus

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.
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pone.0116578.g003: 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.

Mentions: Plots of initial velocity versus NAD concentration were hyperbolic, consistent with Michaelis-Menten kinetics (Fig. 3 and Table 5). The KmNAD values vary from 139 ± 14 to 2113 ± 285 μM. A slight inhibition by excess of NAD was only observed for IMPDHbt (Fig. 3A). Concerning the dependence of activity as a function of IMP concentration (Table 6 and Fig. 4), IMPDHbt, IMPDHba, IMPDHkp and IMPDHsa (class II IMPDHs) exhibited again Michaelis-Menten kinetics. Our kinetic analysis of IMPDHba was slightly different (lower affinity for both substrates) from the work of Makowska-Grzyska et al. [23]. Remarkably, the plots of activity of class I IMPDHs (IMPDHlpp, IMPDHnm and IMPDHpa) as a function of IMP concentration were sigmoidal. However the nH values did not exceed 1.6 and the affinity for IMP was low (K0,5 around 300 μM for IMPDHlpp and IMPDHnm, and 1800 μM for IMPDHpa) compared to other bacterial and non-bacterial IMPDHs [24]. Bacterial IMPDHs were reported to bind IMP efficiently and to have a low affinity for NAD [24]. Our characterization of seven other bacterial IMPDHs indicates that the situation is not that clear-cut.


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

Alexandre T, Raynal B, Rayna B, Munier-Lehmann H - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4338043&req=5

pone.0116578.g003: 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.
Mentions: Plots of initial velocity versus NAD concentration were hyperbolic, consistent with Michaelis-Menten kinetics (Fig. 3 and Table 5). The KmNAD values vary from 139 ± 14 to 2113 ± 285 μM. A slight inhibition by excess of NAD was only observed for IMPDHbt (Fig. 3A). Concerning the dependence of activity as a function of IMP concentration (Table 6 and Fig. 4), IMPDHbt, IMPDHba, IMPDHkp and IMPDHsa (class II IMPDHs) exhibited again Michaelis-Menten kinetics. Our kinetic analysis of IMPDHba was slightly different (lower affinity for both substrates) from the work of Makowska-Grzyska et al. [23]. Remarkably, the plots of activity of class I IMPDHs (IMPDHlpp, IMPDHnm and IMPDHpa) as a function of IMP concentration were sigmoidal. However the nH values did not exceed 1.6 and the affinity for IMP was low (K0,5 around 300 μM for IMPDHlpp and IMPDHnm, and 1800 μM for IMPDHpa) compared to other bacterial and non-bacterial IMPDHs [24]. Bacterial IMPDHs were reported to bind IMP efficiently and to have a low affinity for NAD [24]. Our characterization of seven other bacterial IMPDHs indicates that the situation is not that clear-cut.

Bottom Line: This study led to a classification of bacterial IMPDHs according to the regulation of their catalytic properties and their quaternary structures.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.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Chimie et Biocatalyse, Département de Biologie Structurale et Chimie, 28 rue du Dr Roux, F-75015, Paris, France; Centre Nationale de la Recherche Scientifique, Unité Mixte de Recherche 3523, F-75015, Paris, France; Université Paris Diderot, Sorbonne Paris Cité, F-75205, Paris, France.

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.

Show MeSH
Related in: MedlinePlus