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Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel.

Dou TY, Luan HW, Ge GB, Dong MM, Zou HF, He YQ, Cui P, Wang JY, Hao DC, Yang SL, Yang L - Sci Rep (2015)

Bottom Line: This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones.The key catalytic subunit responsible for the β-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family.The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.

ABSTRACT
Cellulosome is a kind of multienzyme complex that displays high activity, selectivity, and stability. Here, we report a novel, non-cellulolytic, cellulosome-like multienzyme complex that produced by the Cellulosimicrobium cellulans wild-type strain F16 isolated from soil microflora. This multienzyme complex, with excellent catalytic efficiency of kcat 13.2 s(-1) to remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (10-DAXP), has an outstanding tolerance against organic solvents and an excellent general stability, with the long half-life of 214 hours. This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones. The key catalytic subunit responsible for the β-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family. The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

No MeSH data available.


Related in: MedlinePlus

Basic Structures of the Key Subunit: the Hypothetical Protein M768_06655.(a) Primary structure and the corresponding secondary structure predicted by PredictProtein, cartoons were drawn manually according to the prediction, helix—alpha helix, arrow—beta sheet, line-loop structures. (b) and (c), pie chart showing predicted secondary structure composition and solvent accessibility; (d) Protein families.
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f6: Basic Structures of the Key Subunit: the Hypothetical Protein M768_06655.(a) Primary structure and the corresponding secondary structure predicted by PredictProtein, cartoons were drawn manually according to the prediction, helix—alpha helix, arrow—beta sheet, line-loop structures. (b) and (c), pie chart showing predicted secondary structure composition and solvent accessibility; (d) Protein families.

Mentions: To determine the basic structures of these enzymes, three purified enzymes, Xyl_I, Xyl_IV, and Xyl_S, were initially analyzed by SDS-PAGE. The results showed that Xyl_I was actually an enormous multiprotein complex composed of at least 26 subunits, whereas both Xyl_IV and the purified subunit Xyl_S were identified to be a 130 kDa subunit (Fig. 5). Combined with the MALDI-TOF results (Supplementary Fig. 4b,c), these data suggest that these two enzymes are monomeric proteins. To further reveal their primary structures, Xyl_I and Xyl_S were digested by trypsin directly, and then analyzed using the AB SCIEX TripleTOF® 5600 system. The partially purified component Xyl_IV was firstly separated by an 8% native-PAGE gel. The active band, shown by in gel staining using a synthesized fluorescent probe MUX, was cut, in-gel digested by trypsin, and analyzed using the same LC-MS/MS system. The data were then searched against the translated protein sequences from the WGS data. The results showed that, 1) 27 proteins were identified to be subunits of the nanoscale biocatalyst Xyl_I. Further functional predictions showed that they are glucanases, xylanases, mannases, peptidases, and laccases, i.e., mostly hemicellulases. 2) Interestingly, both Xyl_S and the cut-from-gel component Xyl_IV were identified as the hypothetical protein M768_06655, with the C-terminal ~500 aa missing (Fig. 5). However, the full-length version of this protein could be found from the proteomic results of Xyl_I, with a high coverage of 89.7%. Protein domain and family analysis by Pfam18 showed that this hypothetical protein had five significant Pfam-A matches: ThuA (PF06283), GSDH (PF07995), PKD (PF00801), CBM_6 (PF03422), and PKD (PF00801), from the N terminus to the C terminus (Fig. 6d). For further verification and identification of the subunits comprising Xyl_I, the protein bands separated by 10% SDS-PAGE were cut from the gel, in-gel digested by trypsin, analyzed by LTQ LC-MS/MS, and then searched against the translated genome database. The results showed that the hypothetical protein M768_06655 was at the position of ~200 kDa on the gel (Fig. 5). This result validated that Xyl_I contains a full length version of this protein, and both that Xyl_IV and Xyl_S were truncated versions, with the C-terminal approximately 500 aa missing.


Functional and structural properties of a novel cellulosome-like multienzyme complex: efficient glycoside hydrolysis of water-insoluble 7-xylosyl-10-deacetylpaclitaxel.

Dou TY, Luan HW, Ge GB, Dong MM, Zou HF, He YQ, Cui P, Wang JY, Hao DC, Yang SL, Yang L - Sci Rep (2015)

Basic Structures of the Key Subunit: the Hypothetical Protein M768_06655.(a) Primary structure and the corresponding secondary structure predicted by PredictProtein, cartoons were drawn manually according to the prediction, helix—alpha helix, arrow—beta sheet, line-loop structures. (b) and (c), pie chart showing predicted secondary structure composition and solvent accessibility; (d) Protein families.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562250&req=5

f6: Basic Structures of the Key Subunit: the Hypothetical Protein M768_06655.(a) Primary structure and the corresponding secondary structure predicted by PredictProtein, cartoons were drawn manually according to the prediction, helix—alpha helix, arrow—beta sheet, line-loop structures. (b) and (c), pie chart showing predicted secondary structure composition and solvent accessibility; (d) Protein families.
Mentions: To determine the basic structures of these enzymes, three purified enzymes, Xyl_I, Xyl_IV, and Xyl_S, were initially analyzed by SDS-PAGE. The results showed that Xyl_I was actually an enormous multiprotein complex composed of at least 26 subunits, whereas both Xyl_IV and the purified subunit Xyl_S were identified to be a 130 kDa subunit (Fig. 5). Combined with the MALDI-TOF results (Supplementary Fig. 4b,c), these data suggest that these two enzymes are monomeric proteins. To further reveal their primary structures, Xyl_I and Xyl_S were digested by trypsin directly, and then analyzed using the AB SCIEX TripleTOF® 5600 system. The partially purified component Xyl_IV was firstly separated by an 8% native-PAGE gel. The active band, shown by in gel staining using a synthesized fluorescent probe MUX, was cut, in-gel digested by trypsin, and analyzed using the same LC-MS/MS system. The data were then searched against the translated protein sequences from the WGS data. The results showed that, 1) 27 proteins were identified to be subunits of the nanoscale biocatalyst Xyl_I. Further functional predictions showed that they are glucanases, xylanases, mannases, peptidases, and laccases, i.e., mostly hemicellulases. 2) Interestingly, both Xyl_S and the cut-from-gel component Xyl_IV were identified as the hypothetical protein M768_06655, with the C-terminal ~500 aa missing (Fig. 5). However, the full-length version of this protein could be found from the proteomic results of Xyl_I, with a high coverage of 89.7%. Protein domain and family analysis by Pfam18 showed that this hypothetical protein had five significant Pfam-A matches: ThuA (PF06283), GSDH (PF07995), PKD (PF00801), CBM_6 (PF03422), and PKD (PF00801), from the N terminus to the C terminus (Fig. 6d). For further verification and identification of the subunits comprising Xyl_I, the protein bands separated by 10% SDS-PAGE were cut from the gel, in-gel digested by trypsin, analyzed by LTQ LC-MS/MS, and then searched against the translated genome database. The results showed that the hypothetical protein M768_06655 was at the position of ~200 kDa on the gel (Fig. 5). This result validated that Xyl_I contains a full length version of this protein, and both that Xyl_IV and Xyl_S were truncated versions, with the C-terminal approximately 500 aa missing.

Bottom Line: This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones.The key catalytic subunit responsible for the β-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family.The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.

ABSTRACT
Cellulosome is a kind of multienzyme complex that displays high activity, selectivity, and stability. Here, we report a novel, non-cellulolytic, cellulosome-like multienzyme complex that produced by the Cellulosimicrobium cellulans wild-type strain F16 isolated from soil microflora. This multienzyme complex, with excellent catalytic efficiency of kcat 13.2 s(-1) to remove the C-7 xylosyl group from 7-xylosyl-10-deacetylpaclitaxel (10-DAXP), has an outstanding tolerance against organic solvents and an excellent general stability, with the long half-life of 214 hours. This cellulosome-like multienzyme complex has a novel structure distinct from the well-documented ones. The key catalytic subunit responsible for the β-xylosidase activity against 10-DAXP is identified to be a novel protein, indicating a new glycoside hydrolase (GH) family. The pioneering work described here offers a novel nanoscale biocatalyst for the production of biofuels and chemicals from renewable plant-based natural resources.

No MeSH data available.


Related in: MedlinePlus