| Full text | |
| Author(s): |
Calzado, Felipe
;
Prates, Erica T.
;
Goncalves, Thiago A.
;
Rubio, Marcelo V.
;
Zubieta, Mariane P.
;
Squina, Fabio M.
;
Skaf, Munir S.
;
Damasio, Andre R. L.
Total Authors: 8
|
| Document type: | Journal article |
| Source: | Biotechnology and Bioengineering; v. 113, n. 12, p. 2577-2586, DEC 2016. |
| Web of Science Citations: | 3 |
| Abstract | |
Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and -glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves -glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. (c) 2016 Wiley Periodicals, Inc. (AU) | |
| FAPESP's process: | 13/08293-7 - CCES - Center for Computing in Engineering and Sciences |
| Grantee: | Munir Salomao Skaf |
| Support Opportunities: | Research Grants - Research, Innovation and Dissemination Centers - RIDC |
| FAPESP's process: | 14/06923-6 - Sugar cane biomass recalcitrance: Basic knowledge related to the cell wall construction, pretreatment and enzymatic digestion, applied for the development of innovative biorefinery models |
| Grantee: | Andre Luis Ferraz |
| Support Opportunities: | Program for Research on Bioenergy (BIOEN) - Thematic Grants |
| FAPESP's process: | 12/20549-4 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases |
| Grantee: | André Ricardo de Lima Damasio |
| Support Opportunities: | Program for Research on Bioenergy (BIOEN) - Young Investigators Grants |
| FAPESP's process: | 13/24988-5 - Secretion of heterologous glycoproteins in Aspergillus: effect of glycosylation pattern in functional parameters of glycosyl hydrolases |
| Grantee: | Marcelo Ventura Rubio |
| Support Opportunities: | Scholarships in Brazil - Doctorate (Direct) |
| FAPESP's process: | 14/23051-2 - Comparative analysis of the heterologous protein secretion mechanisms in Aspergillus nidulans |
| Grantee: | Felipe Calzado |
| Support Opportunities: | Scholarships in Brazil - Master |