Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases

Abstract

Background:The role of slow off-rates of processive enzymes acting on recalcitrant polysaccharides is poorly understood. Results: Chitinase variants with high off-rates and weak product binding were inefficient in degradation of recalcitrant chitin. Conclusion: Slow off-rates and strong product binding are required for high efficiency and processivity. Significance: Knowledge of determinants of processivity aids to design better enzymes.

Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens

“…Several studies have reported largely different values of the dissociation rate (30 -33). The k off from the chitin nanowhiskers was 0.012 Ϯ 0.002 s Ϫ1 , whereas the k off from the ␣-chitin after 10 min and 2 h of incubation was 0.0028 Ϯ 0.0003 and 0.0015 Ϯ 0.0005 s Ϫ1 , respectively (32). These values were much lower than those found in the present study and our previous study (37).…”

“…In previous studies, the hydrolytic activity of SmChiA against crystalline chitin was found to decrease after the introduction of an alanine mutation into the aromatic residues inside the substrate binding cleft (W167A, W275A, or F396A). These mutants showed reduced levels of processivity on the crystalline chitin (11,32). On the other hand, interestingly, these mutants were found to outperform SmChiA WT in the hydrolysis of soluble chitin and soluble chitosan (11,12).…”

“…Studies on processivity of the cellulases and chitinases using biochemical methods, such as the fluorescence labeling of the substrate (30, 31) and 14 C-labeled chitin (32), or via the use of biosensors (33) have been performed extensively. However, they often involve complicated procedures and have some limitations.…”

Modeling the activity burst in the initial phase of cellulose hydrolysis by the processive cellobiohydrolase Cel7A

“…A key obstacle for an efficient utilization of lignocellulosic biomass is the substrate recalcitrance, manifested by, among other effects, a rapid slowdown of the enzymatic deconstruction of cellulose within minutes after the initial activity burst phase at low cellulose conversion (Cruys‐Bagger et al, ; Murphy et al, ; Praestgaard et al, ). The origin of the activity burst has been attributed to either the heterogeneous nature of the substrate and the changes of the substrate morphology in the course of hydrolysis (Beckham et al, ; Ganner et al, ; Grethlein, ; Peciulyte, Karlström, Larsson, & Olsson, ), or to the enzyme‐related phenomena, such as inactivation of cellulases due to irreversible nonproductive binding onto the cellulose (Cruys‐Bagger et al, , ; Kostylev & Wilson, ; Kurašin, Kuusk, Kuusk, Sørlie & Väljamäe, ). Although experimental evidence from studies employing either pre‐steady‐state measurements (Cruys‐Bagger et al, ; Olsen, Kari, Borch & Westh, ) or time‐resolved high‐resolution visualization techniques (Igarashi et al, ; Nakamura et al, ) provided significant insights regarding the quantitative understanding of cellobiohydrolase action, many fundamental questions still remain open.…”

“…, or to the enzyme-related phenomena, such as inactivation of cellulases due to irreversible nonproductive binding onto the cellulose Kostylev & Wilson, 2013;Kurašin, Kuusk, Kuusk, Sørlie & Väljamäe, 2015). Although experimental evidence from studies employing either pre-steady-state measurements or time-resolved high-resolution visualization techniques (Igarashi et al, 2011;Nakamura et al, 2014) provided significant insights regarding the quantitative understanding of cellobiohydrolase action, many fundamental questions still remain open.…”