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Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Wildberger P, Aish GA, Jakeman DL, Brecker L, Nidetzky B - Biochem Biophys Rep (2015)

Bottom Line: Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase.Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested.High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed (4) C 1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp(196), Glu(237) and Asp(295)) and from Arg(137). Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe(52) by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

No MeSH data available.


Binding of αGlc 1-P to wild-type and mutated LmSPase characterized by STD-NMR. Each STD effect is the ratio of signal intensities in the STD spectrum and in the reference proton spectrum. STD effects are shown normalized on the largest effect in the sample. (A) 1H NMR αGlc 1-P, (B) 1H NMR D-glucose 1-methylene phosphonate, (C) STD-NMR αGlc 1-P/wild-type LmSPase, (D) STD-NMR αGlc 1-P/F52A, (E) STD-NMR αGlc 1-P/F52A_D295A. (F) shows the molecular docking pose of αGlc 1-P in wild-type LmSPase to illustrate the vicinity of ligand C-H groups to aliphatic and aromatic hydrogens of the enzyme.
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f0010: Binding of αGlc 1-P to wild-type and mutated LmSPase characterized by STD-NMR. Each STD effect is the ratio of signal intensities in the STD spectrum and in the reference proton spectrum. STD effects are shown normalized on the largest effect in the sample. (A) 1H NMR αGlc 1-P, (B) 1H NMR D-glucose 1-methylene phosphonate, (C) STD-NMR αGlc 1-P/wild-type LmSPase, (D) STD-NMR αGlc 1-P/F52A, (E) STD-NMR αGlc 1-P/F52A_D295A. (F) shows the molecular docking pose of αGlc 1-P in wild-type LmSPase to illustrate the vicinity of ligand C-H groups to aliphatic and aromatic hydrogens of the enzyme.

Mentions: Fig. 1 (panel D) shows the best-fit docking pose of the phosphonate analog. The predicted binding mode is totally different from that of αGlc 1-P and the calculated binding free energy is less favorable for the analog (E=−4.6 kcal/mol) as compared to αGlc 1-P (E=−5.6 kcal/mol). Fig. 2 summarizes the results of STD-NMR experiments. Binding of αGlc 1-P to LmSPase resulted in a characteristic pattern of STD effects that involved relatively strong interactions from the hydrogens at C6, C4 and C1. Due to overlap of signals from the MES buffer used, STD effects at C3 were not measurable. Just to note, the STD pattern of αGlc 1-P binding to a bacterial starch/maltodextrin phosphorylase from family GT-35 [31] was completely different from the one seen in LmSPase. Proximity of C-H hydrogens from αGlc 1-P to aliphatic or aromatic C–H hydrogens in LmSPase appears to explain the observable pattern of STD effects (Fig. 2). The d-glucose 1-methylene phosphonate did not give measurable STD effects on incubation with LmSPase, thus supporting the notion that binding of the phosphonate to the enzyme was very weak in comparison to the binding of αGlc 1-P.


Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase.

Wildberger P, Aish GA, Jakeman DL, Brecker L, Nidetzky B - Biochem Biophys Rep (2015)

Binding of αGlc 1-P to wild-type and mutated LmSPase characterized by STD-NMR. Each STD effect is the ratio of signal intensities in the STD spectrum and in the reference proton spectrum. STD effects are shown normalized on the largest effect in the sample. (A) 1H NMR αGlc 1-P, (B) 1H NMR D-glucose 1-methylene phosphonate, (C) STD-NMR αGlc 1-P/wild-type LmSPase, (D) STD-NMR αGlc 1-P/F52A, (E) STD-NMR αGlc 1-P/F52A_D295A. (F) shows the molecular docking pose of αGlc 1-P in wild-type LmSPase to illustrate the vicinity of ligand C-H groups to aliphatic and aromatic hydrogens of the enzyme.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4554294&req=5

f0010: Binding of αGlc 1-P to wild-type and mutated LmSPase characterized by STD-NMR. Each STD effect is the ratio of signal intensities in the STD spectrum and in the reference proton spectrum. STD effects are shown normalized on the largest effect in the sample. (A) 1H NMR αGlc 1-P, (B) 1H NMR D-glucose 1-methylene phosphonate, (C) STD-NMR αGlc 1-P/wild-type LmSPase, (D) STD-NMR αGlc 1-P/F52A, (E) STD-NMR αGlc 1-P/F52A_D295A. (F) shows the molecular docking pose of αGlc 1-P in wild-type LmSPase to illustrate the vicinity of ligand C-H groups to aliphatic and aromatic hydrogens of the enzyme.
Mentions: Fig. 1 (panel D) shows the best-fit docking pose of the phosphonate analog. The predicted binding mode is totally different from that of αGlc 1-P and the calculated binding free energy is less favorable for the analog (E=−4.6 kcal/mol) as compared to αGlc 1-P (E=−5.6 kcal/mol). Fig. 2 summarizes the results of STD-NMR experiments. Binding of αGlc 1-P to LmSPase resulted in a characteristic pattern of STD effects that involved relatively strong interactions from the hydrogens at C6, C4 and C1. Due to overlap of signals from the MES buffer used, STD effects at C3 were not measurable. Just to note, the STD pattern of αGlc 1-P binding to a bacterial starch/maltodextrin phosphorylase from family GT-35 [31] was completely different from the one seen in LmSPase. Proximity of C-H hydrogens from αGlc 1-P to aliphatic or aromatic C–H hydrogens in LmSPase appears to explain the observable pattern of STD effects (Fig. 2). The d-glucose 1-methylene phosphonate did not give measurable STD effects on incubation with LmSPase, thus supporting the notion that binding of the phosphonate to the enzyme was very weak in comparison to the binding of αGlc 1-P.

Bottom Line: Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase.Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested.High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, NAWI Graz, Petersgasse 12, A-8010 Graz, Austria.

ABSTRACT

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed (4) C 1 chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme׳s catalytic triad (Asp(196), Glu(237) and Asp(295)) and from Arg(137). Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe(52), a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe(52) by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp(196)) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.

No MeSH data available.