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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.Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

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.

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Formation of E-XMP* during the GMPR reaction. ESI mass spectra of EcGMPR incubated with GMP alone (bottom trace) and with GMP and NADP+ (top trace). Black asterisks (*) indicate EcGMPR that has likely undergone oxidation. EcGMPR observed: 37665 Da (theoretical: 37665 Da). E-XMP* observed: 38012 Da (theoretical: 38010 Da).
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Figure 2: Formation of E-XMP* during the GMPR reaction. ESI mass spectra of EcGMPR incubated with GMP alone (bottom trace) and with GMP and NADP+ (top trace). Black asterisks (*) indicate EcGMPR that has likely undergone oxidation. EcGMPR observed: 37665 Da (theoretical: 37665 Da). E-XMP* observed: 38012 Da (theoretical: 38010 Da).

Mentions: Although the GMPR reaction is presumed to proceed via E-XMP*, this mechanism had not been experimentally verified. Since hydride transfer was slow, E-XMP* should accumulate during the steady-state phase of the reaction. When the reaction was performed with 8-[14C]-GMP and NADPH, radioactivity co-precipitated with enzyme (Supplementary Figure 3). No radioactivity was associated with the enzyme at the completion of the reaction, as expected if a transient intermediate was formed. Approximately 15–20% of the enzyme accumulated as E-XMP* during the steady state. Similar amounts of radioactivity were also trapped in the presence of NADP+. Mass spectrometry revealed that a new peak was formed in the presence of NADP+ that was 347 Da heavier than the apoenzyme, as expected for E-XMP* (Figure 2). Also as expected, no E-XMP* formed when GMPR was incubated with GMP in the absence of NADPH and NADP+ (Figure 2). Therefore the deamination reaction only occurs in the presence of cofactor, although the redox state of the cofactor was not important.


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)

Formation of E-XMP* during the GMPR reaction. ESI mass spectra of EcGMPR incubated with GMP alone (bottom trace) and with GMP and NADP+ (top trace). Black asterisks (*) indicate EcGMPR that has likely undergone oxidation. EcGMPR observed: 37665 Da (theoretical: 37665 Da). E-XMP* observed: 38012 Da (theoretical: 38010 Da).
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Related In: Results  -  Collection

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Figure 2: Formation of E-XMP* during the GMPR reaction. ESI mass spectra of EcGMPR incubated with GMP alone (bottom trace) and with GMP and NADP+ (top trace). Black asterisks (*) indicate EcGMPR that has likely undergone oxidation. EcGMPR observed: 37665 Da (theoretical: 37665 Da). E-XMP* observed: 38012 Da (theoretical: 38010 Da).
Mentions: Although the GMPR reaction is presumed to proceed via E-XMP*, this mechanism had not been experimentally verified. Since hydride transfer was slow, E-XMP* should accumulate during the steady-state phase of the reaction. When the reaction was performed with 8-[14C]-GMP and NADPH, radioactivity co-precipitated with enzyme (Supplementary Figure 3). No radioactivity was associated with the enzyme at the completion of the reaction, as expected if a transient intermediate was formed. Approximately 15–20% of the enzyme accumulated as E-XMP* during the steady state. Similar amounts of radioactivity were also trapped in the presence of NADP+. Mass spectrometry revealed that a new peak was formed in the presence of NADP+ that was 347 Da heavier than the apoenzyme, as expected for E-XMP* (Figure 2). Also as expected, no E-XMP* formed when GMPR was incubated with GMP in the absence of NADPH and NADP+ (Figure 2). Therefore the deamination reaction only occurs in the presence of cofactor, although the redox state of the cofactor was not important.

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.Remarkably, the cofactor is part of the catalytic machinery that activates ammonia.

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