Aug 2026· International Journal of Biological Macromolecules· Vol 381, pp.
154004
· 0 citations· 53 references
Medicine
TL;DR
It is suggested that Ca2+ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain, modulating CBM flexibility and its orientation relative to the catalytic domain in the GH26 family.
Abstract
β-Mannanases are involved in the hydrolysis of mannan, a major component of hemicellulose. The genome of Glutamicibacter halophytocola sp., an endophytic bacterium isolated from Crithmum maritimum, a halophilic plant from the Island of Crete, encodes a putatively secreted β-mannanase (Gh_GH26) belonging to glycoside hydrolase 26 family (GH26). This sequence occupies a poorly explored region of the GH26 sequence space. Gh_GH26 consists of three domains, including a C-terminal carbohydrate-binding module 23 (CBM23), which is uncommon among GH26s and has a peculiar amino acidic composition at the -3/-4 substrate subsites. Gh_GH26 displays halophilic behavior, retaining activity up to 2.5 M NaCl, and exhibits endo-mannanase activity. Functional analyses combined with structural predictions suggest that Gh_GH26 coordinates Ca2+ ions through a single binding site located within the CBM23 domain. The residues involved in Ca2+ binding are conserved in the CBMs of other characterized GH26s, despite their low similarity with Gh_GH26. Site-directed mutagenesis shows that Ca2+ ions contribute to both enzyme activity and stability. These results suggest that Ca2+ may play an evolutionary conserved activation role, modulating CBM flexibility and its orientation relative to the catalytic domain. Overall, this work expands our understanding of the functional and structural diversity of the GH26 family, highlighting its potential for application in the degradation of mannan-containing biomass under saline conditions.
Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.
Sheng-Chia Chen, Po-Chih Kuo, Wen-Ming Chen et al.· Journal of Agricultural and...· 0 citations
This work offers the first molecular insight into P. eryngii xylanases, highlighting PeXyn1 for agro-waste valorization and multiivalent interactions at the CBM1-catalytic interface restrict structural fluctuations, promoting a stable conformation.
Xiaohang Li, Xian-Feng Du, Yan Zhang et al.· Journal of Agricultural and...· 0 citations
Local enrichment of acidic residues on the PlGH3 surface could generate a negative electrostatic potential, which enables adaptation to high-salt and alkaline environments, thereby sustaining the enzyme's catalytic activity under such extreme conditions.
Kaijuan Wu, Ke Guo, Zheng Yu et al.· Applied Biochemistry and Bio...· 0 citations
This study presents the first characterization of a GH3 β-glucosidase from L. buchneri and reveals a non-classical stabilizing effect of EDTA, offering valuable insights for enzyme engineering and biocatalytic applications.
Hui Tang, Jinjian He, Can Li et al.· Metabolites· 0 citations
The CldA enzyme is an unprecedented functional intermediate exhibiting the dual hydrolytic specificity of starch hydrolases and the intramolecular transglycosylation capacity of cyclomaltodextrin glucanotransferases (CGTases) from subfamily 2 of family 13 of glycoside hydrolases (GH13_2). Here, the crystallographic structure of CldA was determined at 1.66 Å resolution. Structural and kinetic studies revealed that the thermophilic CldA has a three-domain ABC architecture similar to that of starch hydrolases from GH13_1, and it contains three calcium-dependent folding centers (Ca+2 1-3) essential for thermostability. However, it simultaneously features nine expanded subsites (-7 to +2) defining the active site cleft of the canonical five-domain ABCDECBM20 CGTases from GH13_2. Structural comparisons revealed three evolutionary adaptations in CldA: (a) the absence of the substrate-guiding DECBM20 domains; (b) an unusual hydrophobic pair, Trp204/Met281, whose hydrophobicity was critical for stabilizing the cyclodextrin (CD) ring at the acceptor subsite +2; and (c) an unexpected hydrogen bond of 2.60 Å between Ser200 at subsite -6 and the key central aromatic residue, Phe216, involved in starch circularization for CD formation. Characterization of two mutants, CldAM281F and CldAS200G, and a five-domain chimera, CldA-DECBM20, provided insights into the boundary between starch hydrolases and CGTases. CldA provides the first experimental structural evidence for a native GH13_2 enzyme where hydrolytic and cyclization activities coexist at a presculpted CGTase active site. Overall, structural and functional analysis of CldA showed that it diverges from canonical GH13_2 CGTases by lacking C-terminal DE domains, shifting its intramolecular transglycosylation specificity toward hydrolysis through an intriguing starch concentration-dependent product-length mechanism.
Beatriz Velazquez-Cruz, Montserrat Romero-Jiménez, Yasel Guerra et al.· The FEBS Journal· 0 citations
A novel type III pullulan hydrolase (PulTS) was identified from the thermophilic bacterium Thermus scotoductus, representing the first bacterial member of this enzyme class. The recombinant PulTS exhibited optimal activity at pH 6.5 and 80 °C, with a specific activity of 46.3 ± 2.4 U/mg toward pullulan. The enzyme hydrolyzed pullulan into maltotriose, panose, maltose, and glucose, and also degraded soluble starch, amylose, amylopectin, dextran, glycogen, and γ-cyclodextrin. PulTS contains sequentially arranged carbohydrate-binding module family 48 (CBM48), CBM34, and glycoside hydrolase family 13 subfamily 20 (GH13_20) catalytic domains, an arrangement previously observed only in archaeal species. Deletion of CBM48 enhanced the thermostability of PulTS by approximately 3-fold, yet the enzyme still retained approximately 69% of its catalytic efficiency toward pullulan. Deletion of CBM34 resulted in no further changes. Affinity gel electrophoresis revealed that CBM48 bound pullulan and, more weakly, soluble starch, while CBM34 exhibited only weak binding ability toward pullulan. The results indicated that CBM48 was the major contributor to substrate binding and enzymatic activity, whereas CBM34 played a minor role in PulTS. This work expands our knowledge of pullulan-hydrolyzing enzymes.
Yi Gao, Zi-Jian Fu, Lan Xu et al.· International Journal of Bio...· 0 citations
We use cookies to run the site and, with your consent, for analytics and to show ads.
See our Cookie Policy.
