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Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

Patton GC, Stenmark P, Gollapalli DR, Sevastik R, Kursula P, Flodin S, Schuler H, Swales CT, Eklund H, Himo F, Nordlund P, Hedstrom L - Nat. Chem. Biol. (2011)

Bottom Line: Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands.The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP.Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

ABSTRACT
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

Show MeSH
The reactions catalyzed by IMPDH and GMPR. (a) Purine nucleotide interconversions. (b) The mechanism of the IMPDH reaction.
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Figure 1: The reactions catalyzed by IMPDH and GMPR. (a) Purine nucleotide interconversions. (b) The mechanism of the IMPDH reaction.

Mentions: The (β/α)8 barrel proteins IMPDH and GMPR present a challenge for the "generalist to specialist" paradigm of enzyme evolution 2. These enzymes have ~30% sequence identity, bind the same ligands with similar affinities and catalyze similar reactions, but with opposing metabolic consequences (Figure 1A; 15). IMPDH catalyzes the conversion of IMP into XMP with the concomitant reduction of NAD+; this is the first committed step in guanine nucleotide biosynthesis. GMPR catalyzes the reduction of GMP to IMP and ammonia with the concomitant oxidation of NADPH. IMPDH is responsible for the expansion of the guanine nucleotide pool required for proliferation 16. In contrast, the guanine nucleotide pool shrinks during differentiation as a result of the expression of GMPR. Thus promiscuous activities would appear to be problematic for these two enzymes – a GMPR activity would be deleterious in IMPDH, and likewise an IMPDH reaction would be unwelcome in GMPR.


Cofactor mobility determines reaction outcome in the IMPDH and GMPR (β-α)8 barrel enzymes.

Patton GC, Stenmark P, Gollapalli DR, Sevastik R, Kursula P, Flodin S, Schuler H, Swales CT, Eklund H, Himo F, Nordlund P, Hedstrom L - Nat. Chem. Biol. (2011)

The reactions catalyzed by IMPDH and GMPR. (a) Purine nucleotide interconversions. (b) The mechanism of the IMPDH reaction.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4552316&req=5

Figure 1: The reactions catalyzed by IMPDH and GMPR. (a) Purine nucleotide interconversions. (b) The mechanism of the IMPDH reaction.
Mentions: The (β/α)8 barrel proteins IMPDH and GMPR present a challenge for the "generalist to specialist" paradigm of enzyme evolution 2. These enzymes have ~30% sequence identity, bind the same ligands with similar affinities and catalyze similar reactions, but with opposing metabolic consequences (Figure 1A; 15). IMPDH catalyzes the conversion of IMP into XMP with the concomitant reduction of NAD+; this is the first committed step in guanine nucleotide biosynthesis. GMPR catalyzes the reduction of GMP to IMP and ammonia with the concomitant oxidation of NADPH. IMPDH is responsible for the expansion of the guanine nucleotide pool required for proliferation 16. In contrast, the guanine nucleotide pool shrinks during differentiation as a result of the expression of GMPR. Thus promiscuous activities would appear to be problematic for these two enzymes – a GMPR activity would be deleterious in IMPDH, and likewise an IMPDH reaction would be unwelcome in GMPR.

Bottom Line: Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands.The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP.Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Brandeis University, Waltham, Massachusetts, USA.

ABSTRACT
Inosine monophosphate dehydrogenase (IMPDH) and guanosine monophosphate reductase (GMPR) belong to the same structural family, share a common set of catalytic residues and bind the same ligands. The structural and mechanistic features that determine reaction outcome in the IMPDH and GMPR family have not been identified. Here we show that the GMPR reaction uses the same intermediate E-XMP* as IMPDH, but in this reaction the intermediate reacts with ammonia instead of water. A single crystal structure of human GMPR type 2 with IMP and NADPH fortuitously captures three different states, each of which mimics a distinct step in the catalytic cycle of GMPR. The cofactor is found in two conformations: an 'in' conformation poised for hydride transfer and an 'out' conformation in which the cofactor is 6 Å from IMP. Mutagenesis along with substrate and cofactor analog experiments demonstrate that the out conformation is required for the deamination of GMP. Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

Show MeSH