Research ArticleBIOCHEMISTRY

“Newton’s cradle” proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography

See allHide authors and affiliations

Science Advances  21 Aug 2015:
Vol. 1, no. 7, e1500263
DOI: 10.1126/sciadv.1500263
  • Fig. 1 Proposed reaction mechanism of inverting cellulase and the close-up views of catalytic site of PcCel45A.

    (A) Reaction scheme of inverting cellulase. The general acid should be protonated and the general base should be deprotonated for the reaction. (B) Comparison of residues around the catalytic centers of HiCel45A (pink) and PcCel45A (green). (C) X-ray omit map of cellopentaose at subsites +1 to +5 of PcCel45A WT at room temperature (2σ level).

  • Fig. 2 Detailed analysis of protonation state of Asn92 by x-ray and neutron crystallographies.

    (A) Determination of orientation and form of Asn92 at room temperature by x-ray diffraction. B-factor values (Å2) are shown above atoms (2FobsFcalc map: 1.5σ in blue, FobsFcalc map: 3.0σ in green and red for positive and negative). (B) Determination of orientation and protonation/deuteration of Asn92 at room temperature by neutron diffraction. B-factor values (Å2) of oxygen and nitrogen atoms are shown above atoms. Ratios of H/D are also shown (2FobsFcalc map: 1.0σ in purple, FobsFcalc map: 2.0σ in green and red for positive and negative).

  • Fig. 3 Proton relay stabilizing the imidic acid state of Asn92.

    (A) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn92 and the amide of Cys96. The 2FobsFcalc (blue) and FobsFcalc (red and green) maps of the x-ray analysis are shown at the 1.0σ and 3.0σ levels, respectively, and 2FobsFcalc (purple) and FobsFcalc (red and green) maps of the neutron analysis are shown at the 1.0σ and 2.0σ levels, respectively. (B) Difference maps calculated without H/D atoms around carbonyl oxygen atoms of Asn105 and amide of Asn105, His107, and Met109. The 2FobsFcalc map of the x-ray analysis is shown at the 1.0σ level, and the FobsFcalc map of the neutron analysis is shown at the 2.0σ level. (C) Proposed mechanism of formation of the imidic acid form of Asn92.

  • Fig. 4 Proton pathway between catalytic residues and expected catalytic mechanism of PcCel45A.

    (A) Difference map calculated without H/D atoms in the proton relay pathway in stereo view. The 2FobsFcalc map (blue) of the x-ray analysis is shown at the 1.0σ level, and the FobsFcalc map (red and green) of the neutron analysis is shown at the 2.0σ level. (B) Proposed Newton’s cradle–like reaction mechanism of PcCel45.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/7/e1500263/DC1

    Table S1. Data collection, phasing, and refinement statistics for first structural determination of PcCel45A.

    Table S2. Statistics of data collection and refinement of the x-ray structure of PcCel45A WT with and without 5 mM cellopentaose at cryogenic temperature.

    Table S3. Statistics of data collection and refinement of the x-ray structure of PcCel45A N92D with and without cellopentaose at cryogenic temperature and PcCel45A N92D unliganded structure at room temperature.

    Table S4. Statistics of data collection and refinement of the x-ray structure of PcCel45A D114N with and without 5 mM cellopentaose at cryogenic temperature.

    Table S5. Statistics of data collection and refinement of the neutron and x-ray structure of PcCel45A WT unliganded.

    Table S6. Statistics of data collection and refinement of the neutron and x-ray structure of PcCel45A WT with 2.5 mM cellopentaose.

    Table S7. Statistics of data collection and refinement of the x-ray structure of PcCel45A N105D.

    Fig. S1. Determination of anomeric forms of products.

    Fig. S2. Structural comparison of PcCel45A with other GH45 enzymes.

    Fig. S3. Activity profile of PcCel45A WT and mutants.

    Fig. S4. Temperature effect on PcCel45A structure.

    Fig. S5. Substrate recognition of PcCel45A WT and mutants.

    Fig. S6. Joint refined structure of PcCel45A WT with 2.5 mM cellopentaose.

    Fig. S7. Neighboring pair residues in the proton relay pathway.

    Fig. S8. Structural and activity profile of PcCel45A N105D mutant.

    Fig. S9. SDS-PAGE and Native-PAGE of PcCel45A WT and mutants.

    Movie S1. The proposed proton relay pathway between Asp92 (general base) and Asn114 (general acid) residues in PcCel45A.

  • Supplementary Materials

    This PDF file includes:

    • Table S1. Data collection, phasing, and refinement statistics for first structural determination of PcCel45A.
    • Table S2. Statistics of data collection and refinement of the x-ray structure of PcCel45A WT with and without 5M cellopentaose at cryogenic temperature.
    • Table S3. Statistics of data collection and refinement of the x-ray structure of PcCel45A N92D with and without cellopentaose at cryogenic temperature and PcCel45A N92D unliganded structure at room temperature.
    • Table S4. Statistics of data collection and refinement of the x-ray structure of PcCel45A D114N with and without 5 mM cellopentaose at cryogenic temperature.
    • Table S5. Statistics of data collection and refinement of the neutron and x-ray structure of PcCel45A WT unliganded.
    • Table S6. Statistics of data collection and refinement of the neutron and x-ray structure of PcCel45A WT with 2.5 mM cellopentaose.
    • Table S7. Statistics of data collection and refinement of the x-ray structure of PcCel45A N105D.
    • Fig. S1. Determination of anomeric forms of products.
    • Fig. S2. Structural comparison of PcCel45A with other GH45 enzymes.
    • Fig. S3. Activity profile of PcCel45A WT and mutants.
    • Fig. S4. Temperature effect on PcCel45A structure.
    • Fig. S5. Substrate recognition of PcCel45A WT and mutants.
    • Fig. S6. Joint refined structure of PcCel45A WT with 2.5 mM cellopentaose.
    • Fig. S7. Neighboring pair residues in the proton relay pathway.
    • Fig. S8. Structural and activity profile of PcCel45A N105D mutant.
    • Fig. S9. SDS-PAGE and Native-PAGE of PcCel45A WT and mutants.
    • Legend for movie S1

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.m4v format). The proposed proton relay pathway between Asp92 (general base) and Asn114 (general acid) residues in PcCel45A.

    Files in this Data Supplement:

Navigate This Article