The structure of the AliC GH13 α-amylase from <i>Alicyclobacillus</i> sp. reveals the accommodation of starch branching points in the α-amylase family

Agirre, Jon; Moroz, Olga V.; Meier, Sebastián; Brask, Jesper; Munch, Astrid; Hoff, Tine; Andersen, Carsten; Wilson, K.S.; Davies, G.J.

doi:10.1107/s2059798318014900

Cited by 22 publications

(14 citation statements)

References 37 publications

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“…Inspection of other GH13 enzymes in complex with Acarbose, which is a tetrasaccharide possessing an N -glycosidic linkage that functions as an inhibitor of α-amylases. Inspection of available GH13 enzymes in complex with Acarbose or Acarbose-derived oligosaccharides (accession codes: 4B9Z, 4E2O, 6GXV, and 6LGE) show that the N -glucosyl moieties in these structures maintain a chair conformation with C(2)–OH and C(3)–OH in equatorial positions 64 – 67 . The difference Sco GlgE1-V279S/ 8 could be due to a low interconversion barrier for 2 H 3 to 3 H 2 in compound 8 .…”

Section: Resultsmentioning

confidence: 99%

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Jayasinghe

Thanvi

et al. 2021

Sci Rep

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Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme family. The results obtained from the dose–response experiments show that 8 at a concentration of 1000 µM reduced the enzyme activity of Sco GlgE1-V279S by 65%. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.83 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)–OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)–OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

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Section: Resultsmentioning

confidence: 99%

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Jayasinghe

Thanvi

et al. 2021

Sci Rep

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“…This difference is likely due to the enzyme/acarbose complex resembling the Michaelis complex rather than mimicking a reaction intermediate as we propose for compound 8. [61][62][63][64] The ammonium moieties in both compounds form polar interactions with the side chain of the General acid/base, Glu 423. This interaction is consistent with the role of Glu 423 in protonating the OH leaving group during the rst catalytic step and is likely stabilizing the complex due to the charge complementary between Glu 423 and the ammonium moieties.…”

Section: Kozikowski Et Al Reported That Hydrogenation Of Substitutedmentioning

confidence: 99%

Stereoselective Synthesis of a 4-⍺-Glucoside of Valienamine, Inhibition, and X-Ray Structural Studies Involving a Bacterial Glycoside Hydrolase-Like Enzyme of The GH13 Family

Jayasinghe

Thanvi

et al. 2021

Preprint

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Glycoside hydrolases (GH) are a large family of hydrolytic enzymes found in all domains of life. As such, they control a plethora of normal and pathogenic biological functions. Thus, understanding selective inhibition of GH enzymes at the atomic level can lead to the identification of new classes of therapeutics. In these studies, we identified a 4-⍺-glucoside of valienamine (8) as an inhibitor (IC50 of 484 ± 51 µM) of Streptomyces coelicolor (Sco) GlgE1-V279S which belongs to the GH13 Carbohydrate Active EnZyme (CAZy) family. The synthetic route to 8 and a closely related 4-⍺-glucoside of validamine (7) was achieved starting from readily available D-maltose. A key step in the synthesis was a chelation-controlled addition of vinylmagnesium bromide to a maltose-derived enone intermediate. X-ray structures of both 7 and 8 in complex with Sco GlgE1-V279S were solved to resolutions of 1.75 and 1.78 Å, respectively. Structural analysis revealed the valienamine derivative 8 binds the enzyme in an E2 conformation for the cyclohexene fragment. Also, the cyclohexene fragment shows a new hydrogen-bonding contact from the pseudo-diaxial C(3)-OH to the catalytic nucleophile Asp 394 at the enzyme active site. Asp 394, in fact, forms a bidentate interaction with both the C(3)-OH and C(7)-OH of the inhibitor. In contrast, compound 7 disrupts the catalytic sidechain interaction network of Sco GlgE1-V279S via steric interactions resulting in a conformation change in Asp 394. These findings will have implications for the design other aminocarbasugar-based GH13-inhibitors and will be useful for identifying more potent and selective inhibitors.

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“…BHA [46] and Alicyclobacillus sp. 18711 (AliC) [47] amylases have a glucose molecule bound near the ring of a tyrosine corresponding to Y358 (in BliAmy), which is an absolutely conserved residue in GH13_5 (Table 3). All ligands discussed have stacking interactions with Y358.…”

Section: Presence Of the Bliamy Sbs In Other Gh13_5 Amylasesmentioning

confidence: 99%

“…In GH13_5 glycosidases, six surface binding sites distinct from the SBS found in BliAmy are observed. A major docking platform is present in amylases AmyG6 (PDB 2D3N) [44,59], BHA (2GJP) [46], AliC (6GXV) [47], Halothermothrix orenii (AmyB, PDB 3BC9) [63] (69, 72, 64 and 44% seq, identity respectively) and in other enzymes mentioned in Table 2 with involvement of two conserved tryptophan residues (W138/ W165 in BliAmy). In AmyG6 W140 and W167 stack with the glucose molecules at subsites −5 and −6 of the active site (Fig.…”

Section: Other Surface Binding Sites In Gh13_5 Glycosidasesmentioning

confidence: 99%

“…identity) has a similar 3-domain structure and an SBS near another conserved tryptophan of the (β/α) 8 barrel domain (Fig. 3B) [47]. However, in BliAmy W184, required for the integrity of the metal binding cage, is also involved in crystal contacts with an arginine side chain of a symmetry related molecule and this site is therefore not available for saccharide binding.…”

Section: Other Surface Binding Sites In Gh13_5 Glycosidasesmentioning

confidence: 99%

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Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

Božić

Rozeboom

Lončar³

et al. 2020

International Journal of Biological Macromolecules

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Bacillus paralicheniformis (BliAmy), belonging to GH13_5 subfamily of glycoside hydrolases, was proven to be a highly efficient raw starch digesting enzyme. The ability of some α-amylases to hydrolyze raw starch is related to the existence of surface binding sites (SBSs) for polysaccharides that can be distant from the active site. Crystallographic studies performed on BliAmy in the apo form and of enzyme bound with different oligosaccharides and oligosaccharide precursors revealed binding of these ligands to one SBS with two amino acids F257 and Y358 mainly involved in complex formation. The role of this SBS in starch binding and degradation was probed by designing enzyme variants mutated in this region (F257A and Y358A). Kinetic studies with different substrates show that starch binding through the SBS is disrupted in the mutants and that F257 and Y358 contributed cumulatively to binding and hydrolysis. Mutation of both sites (F257A/Y358A) resulted in a 5-fold lower efficacy with raw starch as substrate and at least 5.5-fold weaker binding compared to the wild type BliAmy, suggesting that the ability of BliAmy to hydrolyze raw starch with high efficiency is related to the level of its adsorption onto starch granules.

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The structure of the AliC GH13 α-amylase from Alicyclobacillus sp. reveals the accommodation of starch branching points in the α-amylase family

Abstract: In the light of NMR data on product profiles, the structure of an Alicyclobacillus sp. CAZy family GH13 α-amylase highlights the accommodation of branch points in the α-amylase active centre.

Cited by 22 publications

References 37 publications

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Stereoselective synthesis of a 4-⍺-glucoside of valienamine and its X-ray structure in complex with Streptomyces coelicolor GlgE1-V279S

Stereoselective Synthesis of a 4-⍺-Glucoside of Valienamine, Inhibition, and X-Ray Structural Studies Involving a Bacterial Glycoside Hydrolase-Like Enzyme of The GH13 Family

Characterization of the starch surface binding site on Bacillus paralicheniformis α-amylase

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