Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases.

Content Provider	Europe PMC
Author	Nakamura, Akihiko Ishiwata, Daiki Visootsat, Akasit Uchiyama, Taku Mizutani, Kenji Kaneko, Satoshi Murata, Takeshi Igarashi, Kiyohiko Iino, Ryota
Copyright Year	2020
Abstract	Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD.
ISSN	00219258
Volume Number	295
PubMed Central reference number	PMC7586223
Issue Number	43
PubMed reference number	32816991
Journal	The Journal of Biological Chemistry [J. Biol. Chem]
e-ISSN	1083351X
DOI	10.1074/jbc.RA120.014792
Language	English
Publisher	American Society for Biochemistry and Molecular Biology
Publisher Date	2020-08-18
Publisher Place	U.S.A.
Access Restriction	Open
Rights License	Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license. © 2020 Nakamura et al.
Subject Keyword	cellulase cellulose single-molecule observation Cellulomonas fimi carbohydrate-binding protein single-molecule biophysics microscopic imaging molecular imaging glycoside hydrolase processivity glycoside hydrolase family 6 Trichoderma reesei
Content Type	Text
Resource Type	Article
Subject	Cell Biology Molecular Biology Biochemistry