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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.

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NADPH has two conformations in the E•IMP complex of hGMPR2. (a) The cofactor binding site is located in different regions in IMPDH and GMPR. The NAD analog tiazofurin adenine dinucleotide (pink) is shown bound to IMPDH (brown; 1LRT 17). NADPH (green) is shown in the "in" conformation of GMPR (blue, subunits A and D). (b) The interactions of the 2'-phosphoadenosine in GMPR. Subunit A is shown in blue, subunit D in slate blue. Residues within 4 Å are shown. Hydrogen bonds are depicted by cyan lines. (c) The "in" conformation of NADPH (subunit C). (d) The "out" conformation of NADPH (subunit E). (e) Interactions of nicotinamide ribotide in the "in" conformation. Residues within 4 Å of NADP are shown (subunit C; the 2'-phosphoadenosine is omitted). (f) Interactions of nicotinamide ribotide in the "out" conformation. Residues within 4 Å of NADP are shown (subunit E; the 2'-phosphoadenosine is omitted). This figure was rendered with Chimera 50.
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Figure 4: NADPH has two conformations in the E•IMP complex of hGMPR2. (a) The cofactor binding site is located in different regions in IMPDH and GMPR. The NAD analog tiazofurin adenine dinucleotide (pink) is shown bound to IMPDH (brown; 1LRT 17). NADPH (green) is shown in the "in" conformation of GMPR (blue, subunits A and D). (b) The interactions of the 2'-phosphoadenosine in GMPR. Subunit A is shown in blue, subunit D in slate blue. Residues within 4 Å are shown. Hydrogen bonds are depicted by cyan lines. (c) The "in" conformation of NADPH (subunit C). (d) The "out" conformation of NADPH (subunit E). (e) Interactions of nicotinamide ribotide in the "in" conformation. Residues within 4 Å of NADP are shown (subunit C; the 2'-phosphoadenosine is omitted). (f) Interactions of nicotinamide ribotide in the "out" conformation. Residues within 4 Å of NADP are shown (subunit E; the 2'-phosphoadenosine is omitted). This figure was rendered with Chimera 50.

Mentions: Curiously, the cofactor binding site of GMPR was located in a different region of the (β/α)8-barrel than in IMPDH (Figure 4a). While the nicotinamide ribotide portion of NADPH had two different positions, the 2'-phosphoadenosine occupied the same place, and interacted with the same residues, in all seven monomers (Figure 4b, c). The adenine ring interacted with residues from the adjacent subunit, forming hydrogen bonds with Ser314 and Thr317. The adenosine was in the syn conformation, in contrast to most NAD(P) binding enzymes (Supplementary Table 3 contains detailed descriptions of the cofactor conformations)29. Segment 25–28, which was disordered in E•IMP, was ordered in the ternary complex; Ser26 and Arg27 interacted with the 2'-phosphate of the NADPH in the adjacent subunit. Segment 279–286 formed the remainder of the 2'-phosphate binding pocket. This observation suggests that cofactor binding activates the E•GMP complex by moving Arg286 from its position protecting GMP, exposing the purine ring for reaction.


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)

NADPH has two conformations in the E•IMP complex of hGMPR2. (a) The cofactor binding site is located in different regions in IMPDH and GMPR. The NAD analog tiazofurin adenine dinucleotide (pink) is shown bound to IMPDH (brown; 1LRT 17). NADPH (green) is shown in the "in" conformation of GMPR (blue, subunits A and D). (b) The interactions of the 2'-phosphoadenosine in GMPR. Subunit A is shown in blue, subunit D in slate blue. Residues within 4 Å are shown. Hydrogen bonds are depicted by cyan lines. (c) The "in" conformation of NADPH (subunit C). (d) The "out" conformation of NADPH (subunit E). (e) Interactions of nicotinamide ribotide in the "in" conformation. Residues within 4 Å of NADP are shown (subunit C; the 2'-phosphoadenosine is omitted). (f) Interactions of nicotinamide ribotide in the "out" conformation. Residues within 4 Å of NADP are shown (subunit E; the 2'-phosphoadenosine is omitted). This figure was rendered with Chimera 50.
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Figure 4: NADPH has two conformations in the E•IMP complex of hGMPR2. (a) The cofactor binding site is located in different regions in IMPDH and GMPR. The NAD analog tiazofurin adenine dinucleotide (pink) is shown bound to IMPDH (brown; 1LRT 17). NADPH (green) is shown in the "in" conformation of GMPR (blue, subunits A and D). (b) The interactions of the 2'-phosphoadenosine in GMPR. Subunit A is shown in blue, subunit D in slate blue. Residues within 4 Å are shown. Hydrogen bonds are depicted by cyan lines. (c) The "in" conformation of NADPH (subunit C). (d) The "out" conformation of NADPH (subunit E). (e) Interactions of nicotinamide ribotide in the "in" conformation. Residues within 4 Å of NADP are shown (subunit C; the 2'-phosphoadenosine is omitted). (f) Interactions of nicotinamide ribotide in the "out" conformation. Residues within 4 Å of NADP are shown (subunit E; the 2'-phosphoadenosine is omitted). This figure was rendered with Chimera 50.
Mentions: Curiously, the cofactor binding site of GMPR was located in a different region of the (β/α)8-barrel than in IMPDH (Figure 4a). While the nicotinamide ribotide portion of NADPH had two different positions, the 2'-phosphoadenosine occupied the same place, and interacted with the same residues, in all seven monomers (Figure 4b, c). The adenine ring interacted with residues from the adjacent subunit, forming hydrogen bonds with Ser314 and Thr317. The adenosine was in the syn conformation, in contrast to most NAD(P) binding enzymes (Supplementary Table 3 contains detailed descriptions of the cofactor conformations)29. Segment 25–28, which was disordered in E•IMP, was ordered in the ternary complex; Ser26 and Arg27 interacted with the 2'-phosphate of the NADPH in the adjacent subunit. Segment 279–286 formed the remainder of the 2'-phosphate binding pocket. This observation suggests that cofactor binding activates the E•GMP complex by moving Arg286 from its position protecting GMP, exposing the purine ring for reaction.

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
Related in: MedlinePlus