Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber.

Area of Science:

• Biochemistry
• Enzymology
• Biotechnology

Background:

• Cellulose, a polymer of beta-1,4 linked glucans, is abundant in biomass but recalcitrant to enzymatic degradation due to hydrogen bonding.
• Cellobiohydrolases (CBHs) are key enzymes that degrade crystalline cellulose.
• This study focuses on two glycosyl hydrolase family 7 CBHs from Aspergillii that degrade crystalline cellulose.

Purpose of the Study:

• To investigate the distinct roles and synergistic interactions of two fungal cellobiohydrolases, Cbh1 and CelD, in the degradation of crystalline cellulose.
• To elucidate the structural and functional differences between Cbh1, possessing a carbohydrate-binding domain (CBD), and CelD, lacking a CBD.

Main Methods:

• Comparative analysis of Cbh1 and CelD structures, including catalytic domains and substrate-binding channels.
• Enzymatic activity assays on crystalline cellulose (Avicel) and a soluble substrate (pNPC).
• Thermal stability and inhibition studies (glucose).
• Enzyme mixture synergy assays on crystalline cellulosic substrates.

Main Results:

• Cbh1 possesses a CBD and a loop that obstructs its catalytic channel, while CelD lacks a CBD and has a more open channel due to a deletion.
• Cbh1 exhibited significantly higher specific activity on Avicel (7.7 U/mg prot) compared to CelD (0.5 U/mg prot), while activity on pNPC was similar.
• Cbh1 showed greater thermal stability and reduced glucose inhibition compared to CelD.
• Enzyme mixtures demonstrated strong synergy when Cbh1 was in molar excess (2:1 or 4:1), with CelD primarily degrading loosened chains and Cbh1 initiating fiber opening.

Conclusions:

• Fungal genomes commonly encode cellobiohydrolases with and without CBDs, both contributing to cellulose degradation.
• Cbh1's ability to bind and open cellulose fibers is crucial for synergistic degradation by CelD.
• The findings suggest a model where Cbh1 acts as a 'loosener' of cellulose fibers, facilitating efficient hydrolysis by CelD.