Molecular basis of substrate recognition and specificity revealed in family 12 glycoside hydrolases

Abstract

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and β-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 β-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. © 2016 Wiley Periodicals, Inc.

Characterization of acidic endoglucanase Cel12A from Gloeophyllum trabeum and its synergistic effects on hydrogen peroxide–acetic acid (HPAC)-pretreated lignocellulose

“…Recombinant GtCel12A expressed from A. niger strain CBS513.88 and py11 had different protein molecular masses, of 29 and 26.1 kDa, respectively [16,19]. Recombinant EGIIIs (Cel12A) of Trichoderma reesei was expressed in heterologous hosts E. coli, Saccharomyces cerevisiae, and Schizosaccharomyces pombe, which likewise showed different molecular masses (25,28, and 29 kDa, respectively) due to N-glycosylation by the different hosts. The molecular mass was decreased to 25 kDa by endoglycosidase H and α-mannosidase treatment [26].…”

“…The GtCel12A had detectable enzyme activities against both β-glucan and xyloglucan. Thus, GtCel12A can be classified into SF 12-1 of the GH12 family [28]. The kinetic parameters of GtCel12A were as follows: V max = 3452 μmol min −1 mg −1 , K m = 13.83 mg mL −1 , and k cat = 1726 s −1 as determined by a Lineweaver-Burk plot.…”

Loop 3 of Fungal Endoglucanases of Glycoside Hydrolase Family 12 Modulates Catalytic Efficiency

“…Sequence analysis indicated that NfEG12A contains three typical loops of GH12 endoglucanases in the catalytic channel. Loops 1 and 2 are recognized as the catalytic loops (10), which are closely related to substrate specificity and catalytic efficiency. Loop 3 locates on the Ϫ1 or ϩ1 subsites and varies a lot in amino acid composition, but its function is unknown.…”

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System

“…Effective xyloglucan-degrading GH systems (recently reviewed by Attia and Brumer) contain exo -acting glycosidases acting as debranching accessory enzymes and endo -acting enzymes (xyloglucanases) that generate short, branched β-(1,4)-glucan oligomers (xyloglucan oligosaccharides). − Exo -acting enzymes are expected to address each of the glycosidic linkages reported in xyloglucan branches, though the reported enzyme diversity falls short of reported branch diversity. , Endo -acting enzymes, taking on both linear and branched β-(1,4)-glucans, are needed to effectively degrade xyloglucan, yet discrimination between their substrate specificities using contemporary methods (primary sequence alignment, structure alignment) is unreliable, rendering comparisons of xyloglucan-degrading systems from different organisms challenging.…”