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Amylose recognition and ring-size determination of amylomaltase

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Science Advances  13 Jan 2017:
Vol. 3, no. 1, e1601386
DOI: 10.1126/sciadv.1601386
  • Fig. 1 Reaction mechanism of TaqAM leading to transglycosylation or hydrolysis.
  • Fig. 2 Cocrystal structure of CA34 bound to TaqAM.

    (A) Overview of the TaqAM fold. In addition to the (β/α)8-barrel, three subdomains (B1, B2, and B3) are present. (B) Representation of the crystallographic dimer bridged by the bound CA34 (shown as blue glycoblock representation). The symmetry-related monomers are shown in red and beige. (C) Binding of CA residues 1 to 16 to one monomer of TaqAM shown in stereo. The corresponding 2mFoDFc electron density around the CA after refinement is contoured at 1σ.

  • Fig. 3 Interactions of CA34 with TaqAM.

    Binding of CA residues 1 to 16 to one monomer of TaqAM. Glc-1 (green) and the preceding residues of the donor site are shown on the left side with the carbon in gray, whereas Glc+1 (red) and the following residues in the acceptor binding sites are on the right side with the carbon in yellow. The 250 loop (amino acids 247 to 255) is in purple, the 370 loop (amino acids 368 to 372) is in dark pink, the 460 loop (amino acids 458 to 472) is in dark blue, F366 (part of the acceptor sugar tong) is in light blue, whereas the remaining molecule is in beige. These loops are assumed to be of importance for AM function (2).

  • Fig. 4 Sugar tongs and succinimide in CA binding.

    (A) Close-up of the acceptor sugar tong formed by F251 and F366. (B) Movement of the 250 loop in response to inhibitor or substrate binding. The unliganded enzyme is in dark green [Protein Data Bank (PDB) ID, 1CWY], the acarbose complex is in purple (PDB ID, 1ESW), and the CA34-bound structure is in beige. The bound CA34 is shown in glycoblocks for better orientation, and the tong-forming residues are depicted in stick representation. (C) The imide formed by D370. The imide and D369 are shown in stick representation with the corresponding 2mFoDFc density contoured at 1.5σ (blue). The hydrogen bond of the imide with O2 of Glc+5 is shown as dashed lines. The remaining hydrogen bond contacts on the acceptor site with D369 and E373 are shown as well. (D) Schematic representation of the imide formation. The imide forms through a nucleophilic attack of the peptide backbone nitrogen on the carboxyl carbon of the preceding aspartate. The subsequent dehydration leads to a stable five-membered ring structure.

  • Fig. 5 CA34 mimics amylose binding in the secondary binding site.

    Close-up of the secondary binding site of AM with bound CA34 shown in blue glycoblocks around Y54. Overlaid in orange is a part of the helix of the crystallized CA26 (PDB ID, 1C58), which resembles the helical structure of V-amylose (35). The two sugar chains have a similar helical conformation, revealing that CA34 in this binding site adopts a conformation, which resembles the low-energy conformation of the free amylose substrate.

  • Fig. 6 Schematic catalytic cycle for CA34 formation catalyzed by AMs.

    (A) Starch amylose binds to AM at the secondary binding site in the native V-helical conformation. This binding site may thus be the site for initial substrate recognition. (B) The chain then protrudes into the substrate-binding crevice until all 16 subsites are filled. (C) After cleavage of the glycosidic bond following nucleophilic attack of D293 in the active site, the nonreducing end folds back into the acceptor site (D), resulting in the formation of cyclic amylose (E). Because of the 10 already occupied donor binding sites, the crevice defines the minimal ring size of the formed CAs, thereby acting as molecular ruler, and the two ends of the 34-glucose-long amylose are ligated at D293 to form CA34. (F) Release of the CA product. “L-250” and “L-460” refer to the 250 loop and 460 loop, respectively.

  • Table 1 Geometric parameter for the carbohydrate units of CA34.

    Torsion angles that deviate by >10° from those observed in V-amylose and distances O3(n)–O2(n + 1) that are too long for a hydrogen bonding interaction are underlined to indicate conformational changes of the CA34 molecule due to binding to AM. Residues −7 to −9, which form the core of the helical region wrapping around Y54, most closely correspond to the V-amylose helix conformation as characterized by the torsion angles ϕ between 91° and 115° as well as ψ between 97° and 131° and by the presence of O3(n)–O2(n + 1) hydrogen bonds as observed in the helical regions of the crystal structure of CA26 (36).

    SugarResidueB factor [Å2]Φ [°] O5(n)−C1(n)−
    O4(n − 1)−C4(n − 1)
    Ψ [°] C1(n)−O4(n − 1)−
    C4(n − 1)−C3(n − 1)
    χ [°] (O5−C5−C6−O6)Distance [Å] O3(n)−
    O2(n + 1)
    1+6117.9111.665.94−66.63.07
    2+544.999.799.9−59.32.94
    3+443.0104.598.1−69.14.04
    4+378.973.890.8−77.14.36
    5+260.560.583.6−55.94.06
    6+143.965.290.161.34.21
    7−163.459.188.349.83.80
    8−271.874.989.129.93.60
    9−3112.781.984.655.64.72
    10−4124.844.572.365.72.26
    11−5148.7141.1121.038.73.56
    12−674.386.683.7163.32.84
    13−776.8114.5116.455.02.85
    14−897.6110.495.450.32.59
    15−9102.6120.5123.842.43.72
    16−10126.184.783.492.24.47
    17−11130.860.967.0148.43.51
  • Table 2 Enzymatic activities of AM and mutants thereof, divided into cyclization, disproportionation, coupling, and hydrolysis.
    AMCyclizationDisproportionationCouplingHydrolysis
    U/mgRelative (%)U/mgRelative (%)U/mgRelative (%)U/mgRelative (%)
    Wild type0.31710011881000.3161000.308100
    Y54G0.05718665.60.190600.10133
    S57R0.003150.40.0010.30.03712
    Y101G0.0113.5139120.0061.90.16453
    +SY250F251+S0.00020.1100.80.0020.60.12541
    D370S0.1695313401130.125400.414134
  • Table 3 Kinetic parameters for the cyclization, disproportionation, and coupling reactions catalyzed by the wild-type and mutated AMs.
    AMCyclizationDisproportionationCoupling
    Km pea starch
    (mM)
    kcat
    (s−1)
    kcat/Km
    (s−1 mM−1)
    Km maltotriose
    (mM)
    kcat
    (s−1)
    kcat/Km
    (s−1 mM−1)
    Km CD25
    (mM)
    kcat
    (s−1)
    kcat/k
    (s−1 mM−1)
    Wild type3.25 × 10−6 ± 4 × 10−72.0 × 10−56.152.48 × 10−2 ± 2 × 10−39.8 × 10−23.952.48 × 10−3 ± 2 × 10−48.2 × 10−33.31
    Y54G1.53 × 10−6 ± 2 × 10−71.2 × 10−60.781.41 × 10−2 ± 1 × 10−31.6 × 10−30.113.74 × 10−3 ± 2 × 10−44.5 × 10−31.20
    S57R2.86 × 10−6 ± 5 × 10−72.1 × 10−80.00731.75 × 10−2 ± 2 × 10−31.0 × 10−40.0066.69 × 10−3 ± 2 × 10−43.0 × 10−50.005
    Y101G1.13 × 10−5 ± 4 × 10−62.0 × 10−70.0182.70 × 10−2 ± 3 × 10−31.0 × 10−20.374.80 × 10−2 ± 9 × 10−25.5 × 10−30.12
    +SY250F251+S5.68 × 10−6 ± 4 × 10−75.1 × 10−60.901.03 × 10−2 ± 1 × 10−36.6 × 10−30.642.86 × 10−3 ± 2 × 10−41.0 × 10−50.004
    D370S5.69 × 10−6 ± 2 × 10−62.8 × 10−54.921.61 × 10−2 ± 5 × 10−38.9 × 10−25.537.27 × 10−3 ± 6 × 10−46.2 × 10−30.85
  • Table 4 Data collection and refinement statistics.

    Values in parentheses refer to the highest-resolution shell.

    TaqAMv (D293A and D395N)
    X-ray sourceBL 14.2 (HZB)
    Wavelength (Å)0.91841
    Space group, unit cell (Å)I422, a = b = 157.6, c =112.8
    Resolution range (Å)37–1.73
    Completeness (%)99.3 (99.9)
    Rmerge0.057 (0.756)
    I/σ(I)16.9 (3.7)
    Wilson B factor (Å2)36
    Redundancy18
    R/Rfree (%)16.40/18.84
    B factor average (Å2)51
      Protein (Å2)48
      Water (Å2)52
      Carbohydrate (Å2)81
    Number of atoms
      Protein4377
      Water443
      Cycloamylose/carbohydrates187
    Ramachandran plot
      Most favored (%)98.0
      Allowed (%)1.2
      Disallowed (%)0.8
    Root mean square deviation
      Bond length (Å)0.005
      Bond angle (°)0.807

Supplementary Materials

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Simulated annealing composite omit difference map of CA34 bound to TaqAM.

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