Kinetic Modeling at Single-Molecule Resolution Elucidates the Mechanisms of Cellulase Synergy

Abstract

The mechanism of synergistic cooperation between cellulases in decomposing cellulose is revealed by resolving the molecular structures of enzymes and substrates via kinetic modeling. The emergence of endo−exo and exo− exo synergy through enzyme−enzyme and enzyme−substrate couplings was investigated with the main exoglucanases (TrCel7A and TrCel6A) and endoglucanase (TrCel7B) of the Trichoderma reesei fungus. The degree of synergy was found to depend on the interplay between two competing effects: (1) enhancement of the complexation rates of exoglucanases with the chain ends created by endoglucanases and (2) surface inhibition of processive exoglucanases by uneven layers of glucan chains on cellulose. We show that the sole effect of TrCel7B in creating more free ends for TrCel7A is insufficient to cause synergistic activity. The combined actions of TrCel7A and TrCel7B produce a rougher substrate surface that subsequently promotes blocking of processing TrCel7A enzymes. This anti-synergy can be counteracted by enhancing the rates of complexation of TrCel7A with the TrCel7B-created chain ends, allowing their synergistic cooperation. Similarly, kinetic simulations show that exo−exo synergy does not occur if TrCel6A and TrCel7A have only opposite specificities in targeting the two ends of glucan chains. Incorporating endo activity into TrCel6A with complexation rate enhancement, however, can lead to synergy with TrCel7A. Therefore, we find that endo−exo and exo−exo synergies may share the same mechanistic origin. The results of work also highlight that resolving molecular configurations in kinetic modeling allows systematic analysis for elucidating the mechanism of interfacial biocatalysis.

Cellular automata modeling depicts degradation of cellulosic material by a cellulase system with single-molecule resolution

“…Figure 2 a additionally shows the so-called hydrophobic faces of the nanofibril, indicating the crystalline surfaces where enzymatic attack takes place [ 19 , 42 ]. A similar modeling concept was used recently to describe the orientation and accessibility of the hydrophobic faces in cellulose nanofibrils [ 30 , 43 ]. Note: the possibility that individual cellulose chains of the hydrophobic face differ in “decrystallization energy” and therefore in accessibility to enzymatic attack, as has been suggested in in-depth modeling studies of the crystalline cellulose structure [ 32 , 44 ], was not considered in our model.…”

The productive cellulase binding capacity of cellulosic substrates

“…For example, endocellulases are expected to increase productive binding site concentrations for exocellulases (Jeoh et al, 2002;Shang and Chu, 2014). For example, endocellulases are expected to increase productive binding site concentrations for exocellulases (Jeoh et al, 2002;Shang and Chu, 2014).…”

“…It has long been surmised that synergy between hydrolytic cellulases is due to the generation of productive binding sites. For example, endocellulases are expected to increase productive binding site concentrations for exocellulases (Jeoh et al, 2002;Shang and Chu, 2014). More recently, oxidative cleavage of cellulose glycosidic bonds by lytic polysaccharide monooxygenase (LPMO) have also been surmised to increase productive binding site concentrations for exocellulases (Eibinger et al, 2014).…”

Cellulase biocatalysis: key influencing factors and mode of action

“…Wang et al (2012) reported that the preferential digestion of amorphous/para-crystlline cellulose by Cel7B could explain the synergy of this enzyme to other two cellulases such as Cel7A and Cel6A (Wang et al 2012). The magnitude of synergism has been depended on the rate of complexation of exoglucanase with the chain end that created by endoglucanase and the inhibition by uneven surface layers of cellulose chain (Shang and Chu 2014). However, other investigations have observed that not all combination of endo-and exo-type enzymes could show effective synergism (Streamer et al 1975;Wood 1975).…”