Abstract
The association of glycosyl hydrolases with catalytically inactive modules is a successful evolutionary strategy that is commonly used by biomass-degrading microorganisms to digest plant cell walls. The presence of accessory domains in these enzymes is associated with properties such as higher catalytic efficiency, extension of the catalytic interface and targeting of the enzyme to the proper substrate. However, the importance of the linker region in the synergistic action of the catalytic and accessory domains remains poorly understood. Thus, this study examined how the inter-domain region affects the structure and function of modular GH5 endoglucanases, by using cellulase 5A from Bacillus subtilis (BsCel5A) as a model. BsCel5A variants featuring linkers with different stiffnesses or sizes were designed and extensively characterized, revealing that changes in flexibility or rigidity in this region differentially affect kinetic behavior. Regarding the linker length, we found that precise inter-domain spacing is required to enable efficient hydrolysis because excessively long or short linkers were equally detrimental to catalysis. Together, these findings identify molecular and structural features that may contribute to the rational design of chimeric and multimodular glycosyl hydrolases.
Highlights
Cellulose, the most abundant biopolymer on earth[1], plays a critical role in the recycling of photosynthetically fixed carbon
Cellulose is primarily broken down through two pathways: one is widespread among aerobic bacteria and fungi and involves only individual glycosyl hydrolases, and the other is restricted to a few anaerobic microorganisms, in which these complementary enzymes form a macromolecular assembly called the cellulosome[4]
To examine the effects of changes in length, chimeric proteins with linkers that were two-fold (L56 – 56 residues long) and four-fold (L104 – 104 residues long) longer than that of wild-type BsCel5A were synthesized on the basis of the sequences of GH family 05 (GH5)-CBM3 cellulases from Paenibacillus genus (Fig. 1B and S1)
Summary
The most abundant biopolymer on earth[1], plays a critical role in the recycling of photosynthetically fixed carbon. Cellulose is primarily broken down through two pathways: one is widespread among aerobic bacteria and fungi and involves only individual glycosyl hydrolases, and the other is restricted to a few anaerobic microorganisms, in which these complementary enzymes form a macromolecular assembly called the cellulosome[4] Between these two main pathways lies an alternative strategy that involves multimodular cellulases harboring different substrate-binding modules and catalytic domains in the same polypeptide. The mechanism of this intermediate strategy was recently described by Brunecky and co-workers, who have characterized the enzyme CelA from the thermophilic bacterium Caldicellulosiruptor bescii. The glycosylation of eukaryotic linkers renders them more resistant to proteolysis and enhances their cellulose-binding affinity[11]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
