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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.


Close-up views of the predicted binding of αGlc 1-P dianion at the catalytic site of sucrose phosphorylase. (A) Wild-type LmSPase (modeled). (B) E232N mutant of BaSPase (PDB entry 2gdu). (C) For reference, αGlc 1-P bound in maltodextrin phosphorylase from E. coli (PDB entry 1l5v). (D) d-Glucose 1-methylene phosphonate (dianion) binding to wild-type LmSPase. (E) F52A mutant. (F) F52A_D295A double mutant. Ligand carbon atoms are colored green, except for panel C where yellow color is used to highlight the different enzyme system. The pyridoxal 5′-phosphate cofactor of maltodextrin phosphorylase is also shown in panel C. Hydrogen bonds (≤3.5 Å) are shown as black-dashed lines. Interactions potentially relevant for catalysis are shown as gray-dashed lines. Distances are given in Å.
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f0005: Close-up views of the predicted binding of αGlc 1-P dianion at the catalytic site of sucrose phosphorylase. (A) Wild-type LmSPase (modeled). (B) E232N mutant of BaSPase (PDB entry 2gdu). (C) For reference, αGlc 1-P bound in maltodextrin phosphorylase from E. coli (PDB entry 1l5v). (D) d-Glucose 1-methylene phosphonate (dianion) binding to wild-type LmSPase. (E) F52A mutant. (F) F52A_D295A double mutant. Ligand carbon atoms are colored green, except for panel C where yellow color is used to highlight the different enzyme system. The pyridoxal 5′-phosphate cofactor of maltodextrin phosphorylase is also shown in panel C. Hydrogen bonds (≤3.5 Å) are shown as black-dashed lines. Interactions potentially relevant for catalysis are shown as gray-dashed lines. Distances are given in Å.

Mentions: Sucrose phosphorylase catalyzes the conversion of sucrose and phosphate into α-d-glucose 1-phosphate (αGlc 1-P) and d-fructose [1,2]. The enzyme belongs to a large family of glycoside hydrolases and transglycosylases (family GH-13) that act on α-glucosidic oligosaccharide and polysaccharide substrates [3,4]. Sucrose phosphorylase presents a unique enzymatic reactivity within family GH-13 (EC 2.4.1.7) that involves utilization of phosphate as the glucosyl acceptor substrate. Mechanistically, as in other enzymes of family GH-13 [4], reaction occurs through a double displacement-like catalytic process that implicates a covalent β-glucosyl enzyme intermediate and therefore proceeds with retention of α-anomeric configuration in the transferred d-glucosyl residue [2,5]. The active-site of sucrose phosphorylase consists of a highly conserved Asp/Glu/Asp triad of residues that fulfill key roles in catalysis, as shown in Scheme 1[2,5–10]. An aromatic residue, a phenylalanine, is also present (Fig. 1) and a catalytic function has only recently been proposed: the π electron cloud of the phenyl sidechain provides selective stabilization of the glucosyl oxocarbenium ion-like species formed in each transition state of the reaction [11]. The aromatic residue is highly conserved among glycoside hydrolases of family GH-13, as pointed out by Wildberger et al. [11], and it is either a Phe or a Tyr. Its function in catalysis is likely to be also conserved across the large family GH-13.


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)

Close-up views of the predicted binding of αGlc 1-P dianion at the catalytic site of sucrose phosphorylase. (A) Wild-type LmSPase (modeled). (B) E232N mutant of BaSPase (PDB entry 2gdu). (C) For reference, αGlc 1-P bound in maltodextrin phosphorylase from E. coli (PDB entry 1l5v). (D) d-Glucose 1-methylene phosphonate (dianion) binding to wild-type LmSPase. (E) F52A mutant. (F) F52A_D295A double mutant. Ligand carbon atoms are colored green, except for panel C where yellow color is used to highlight the different enzyme system. The pyridoxal 5′-phosphate cofactor of maltodextrin phosphorylase is also shown in panel C. Hydrogen bonds (≤3.5 Å) are shown as black-dashed lines. Interactions potentially relevant for catalysis are shown as gray-dashed lines. Distances are given in Å.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4554294&req=5

f0005: Close-up views of the predicted binding of αGlc 1-P dianion at the catalytic site of sucrose phosphorylase. (A) Wild-type LmSPase (modeled). (B) E232N mutant of BaSPase (PDB entry 2gdu). (C) For reference, αGlc 1-P bound in maltodextrin phosphorylase from E. coli (PDB entry 1l5v). (D) d-Glucose 1-methylene phosphonate (dianion) binding to wild-type LmSPase. (E) F52A mutant. (F) F52A_D295A double mutant. Ligand carbon atoms are colored green, except for panel C where yellow color is used to highlight the different enzyme system. The pyridoxal 5′-phosphate cofactor of maltodextrin phosphorylase is also shown in panel C. Hydrogen bonds (≤3.5 Å) are shown as black-dashed lines. Interactions potentially relevant for catalysis are shown as gray-dashed lines. Distances are given in Å.
Mentions: Sucrose phosphorylase catalyzes the conversion of sucrose and phosphate into α-d-glucose 1-phosphate (αGlc 1-P) and d-fructose [1,2]. The enzyme belongs to a large family of glycoside hydrolases and transglycosylases (family GH-13) that act on α-glucosidic oligosaccharide and polysaccharide substrates [3,4]. Sucrose phosphorylase presents a unique enzymatic reactivity within family GH-13 (EC 2.4.1.7) that involves utilization of phosphate as the glucosyl acceptor substrate. Mechanistically, as in other enzymes of family GH-13 [4], reaction occurs through a double displacement-like catalytic process that implicates a covalent β-glucosyl enzyme intermediate and therefore proceeds with retention of α-anomeric configuration in the transferred d-glucosyl residue [2,5]. The active-site of sucrose phosphorylase consists of a highly conserved Asp/Glu/Asp triad of residues that fulfill key roles in catalysis, as shown in Scheme 1[2,5–10]. An aromatic residue, a phenylalanine, is also present (Fig. 1) and a catalytic function has only recently been proposed: the π electron cloud of the phenyl sidechain provides selective stabilization of the glucosyl oxocarbenium ion-like species formed in each transition state of the reaction [11]. The aromatic residue is highly conserved among glycoside hydrolases of family GH-13, as pointed out by Wildberger et al. [11], and it is either a Phe or a Tyr. Its function in catalysis is likely to be also conserved across the large family GH-13.

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.