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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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Reactions of EcGMPR. (a) Formation of E-XMP* from [14C]-GMP and EcGMPR in presence of NADP analogs. Results are the average and standard deviation of two trials. Thio-NADP, thionicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; APAD, acetylpyridine adenine dinucleotide phosphate. (b) EcGMPR catalyzes the reaction of IMP, NADP+ and ammonia to GMP as measured by production of NADPH. (c) Over-expression of EcGMPR can replace IMPDH/GMPS. pET28a, empty vector; pEcIMPDH, expresses wild-type EcIMPDH; pEcGMPRwt, expresses wild-type EcGMPR; pEcGMPRC/A, expresses the Cys186Ala variant; pEcGMPRT/A, expresses the Thr188Ala variant; pEcGMPRE/Q, expresses the Glu289Gln variant. BL21 ΔguaC, which contains a deletion in the gene that encodes GMPR, grows under all conditions as expected. BL21 ΔguaB contains a deletion in the gene that encodes IMPDH. This deletion also attenuates expression of the neighboring gene that encodes GMPS (Supplementary Figure 11a).
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Figure 5: Reactions of EcGMPR. (a) Formation of E-XMP* from [14C]-GMP and EcGMPR in presence of NADP analogs. Results are the average and standard deviation of two trials. Thio-NADP, thionicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; APAD, acetylpyridine adenine dinucleotide phosphate. (b) EcGMPR catalyzes the reaction of IMP, NADP+ and ammonia to GMP as measured by production of NADPH. (c) Over-expression of EcGMPR can replace IMPDH/GMPS. pET28a, empty vector; pEcIMPDH, expresses wild-type EcIMPDH; pEcGMPRwt, expresses wild-type EcGMPR; pEcGMPRC/A, expresses the Cys186Ala variant; pEcGMPRT/A, expresses the Thr188Ala variant; pEcGMPRE/Q, expresses the Glu289Gln variant. BL21 ΔguaC, which contains a deletion in the gene that encodes GMPR, grows under all conditions as expected. BL21 ΔguaB contains a deletion in the gene that encodes IMPDH. This deletion also attenuates expression of the neighboring gene that encodes GMPS (Supplementary Figure 11a).

Mentions: To probe the importance of the cofactor in the deamination reaction, the reaction was performed with acetylpyridine adenine dinucleotide phosphate (APADPH), where the amide group is replaced with a methylketone. No reaction with GMP and APADPH was detected with EcGMPR, in agreement with an investigation of human GMPR (Table 1; 26). Likewise, only a minor amount of E-XMP* was detected when 8-[14C]-GMP was incubated with enzyme and APADP+, and no E-XMP* was formed in the presence of thionicotinamide adenine dinucleotide phosphate or nicotinic acid adenine dinucleotide phosphate (Figure 5a). In contrast, EcGMPR catalyzed the reaction between 2-Cl-IMP and APADPH (Table 1; KM(APADPH) = 11.0 ± 2.0 µM) and kcat of 0.08 ± 0.01 s−1). These observations indicated that the amide group of the cofactor played a crucial role in the deamination of GMP.


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)

Reactions of EcGMPR. (a) Formation of E-XMP* from [14C]-GMP and EcGMPR in presence of NADP analogs. Results are the average and standard deviation of two trials. Thio-NADP, thionicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; APAD, acetylpyridine adenine dinucleotide phosphate. (b) EcGMPR catalyzes the reaction of IMP, NADP+ and ammonia to GMP as measured by production of NADPH. (c) Over-expression of EcGMPR can replace IMPDH/GMPS. pET28a, empty vector; pEcIMPDH, expresses wild-type EcIMPDH; pEcGMPRwt, expresses wild-type EcGMPR; pEcGMPRC/A, expresses the Cys186Ala variant; pEcGMPRT/A, expresses the Thr188Ala variant; pEcGMPRE/Q, expresses the Glu289Gln variant. BL21 ΔguaC, which contains a deletion in the gene that encodes GMPR, grows under all conditions as expected. BL21 ΔguaB contains a deletion in the gene that encodes IMPDH. This deletion also attenuates expression of the neighboring gene that encodes GMPS (Supplementary Figure 11a).
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Figure 5: Reactions of EcGMPR. (a) Formation of E-XMP* from [14C]-GMP and EcGMPR in presence of NADP analogs. Results are the average and standard deviation of two trials. Thio-NADP, thionicotinamide adenine dinucleotide phosphate; NAADP, nicotinic acid adenine dinucleotide phosphate; APAD, acetylpyridine adenine dinucleotide phosphate. (b) EcGMPR catalyzes the reaction of IMP, NADP+ and ammonia to GMP as measured by production of NADPH. (c) Over-expression of EcGMPR can replace IMPDH/GMPS. pET28a, empty vector; pEcIMPDH, expresses wild-type EcIMPDH; pEcGMPRwt, expresses wild-type EcGMPR; pEcGMPRC/A, expresses the Cys186Ala variant; pEcGMPRT/A, expresses the Thr188Ala variant; pEcGMPRE/Q, expresses the Glu289Gln variant. BL21 ΔguaC, which contains a deletion in the gene that encodes GMPR, grows under all conditions as expected. BL21 ΔguaB contains a deletion in the gene that encodes IMPDH. This deletion also attenuates expression of the neighboring gene that encodes GMPS (Supplementary Figure 11a).
Mentions: To probe the importance of the cofactor in the deamination reaction, the reaction was performed with acetylpyridine adenine dinucleotide phosphate (APADPH), where the amide group is replaced with a methylketone. No reaction with GMP and APADPH was detected with EcGMPR, in agreement with an investigation of human GMPR (Table 1; 26). Likewise, only a minor amount of E-XMP* was detected when 8-[14C]-GMP was incubated with enzyme and APADP+, and no E-XMP* was formed in the presence of thionicotinamide adenine dinucleotide phosphate or nicotinic acid adenine dinucleotide phosphate (Figure 5a). In contrast, EcGMPR catalyzed the reaction between 2-Cl-IMP and APADPH (Table 1; KM(APADPH) = 11.0 ± 2.0 µM) and kcat of 0.08 ± 0.01 s−1). These observations indicated that the amide group of the cofactor played a crucial role in the deamination of GMP.

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