Jeppe Kari

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

Background: To elucidate the rate-determining steps of cellobiohydrolase Cel7A from T. reesei, variants with lower substrate affinity were designed. Results: Mutant (W38A) had reduced substrate affinity but a 2-fold increase in the maximum quasi-steady-state rate. Conclusion: Dissociation of stalled TrCel7A is the rate-limiting step in the initial phase of hydrolysis. Significance: This work offers a new perspective for the design of faster cellulases. Cellobiohydrolases are exo-active glycosyl hydrolases that processively convert cellulose to soluble sugars, typically cellobiose. They effectively break down crystalline cellulose and make up a major component in industrial enzyme mixtures used for deconstruction of lignocellulosic biomass. Identification of the rate-limiting step for cellobiohydrolases remains controversial, and recent reports have alternately suggested either association (on-rate) or dissociation (off-rate) as the overall bottleneck. Obviously, this uncertainty hampers both fundamental mechanistic understanding and rational design of enzymes with improved industrial applicability. To elucidate the role of on- and off-rates, respectively, on the overall kinetics, we have expressed a variant in which a tryptophan residue (Trp-38) in the middle of the active tunnel has been replaced with an alanine. This mutation weakens complex formation, and the population of substrate-bound W38A was only about half of the wild type. Nevertheless, the maximal, steady-state rate was twice as high for the variant enzyme. It is argued that these opposite effects on binding and activity can be reconciled if the rate-limiting step is after the catalysis (i.e. in the dissociation process).

Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7)

Background: Substrate interactions in the long tunnel of processive cellulases govern both their catalytic activity and stepwise movement along a cellulose strand. Results: The energetics of enzyme-substrate interactions at different depths of the tunnel are reported. Conclusion: The affinity for the substrate varies strongly through the tunnel. Significance: Quantitative information on interactions is required to understand the complex processive mechanism. Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 105 m−1, and for longer ligands (DP 5–8) this value was ∼107 m−1. Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel.

Inter-domain synergism is required for efficient feeding of cellulose chain into active site of cellobiohydrolase Cel7A.

Structural polysaccharides like cellulose and chitin are abundant and their enzymatic degradation to soluble sugars is an important route in green chemistry. Processive glycoside hydrolases (GHs), like cellobiohydrolase Cel7A of Trichoderma reesei (TrCel7A) are key components of efficient enzyme systems. TrCel7A consists of a catalytic domain (CD) and a smaller carbohydrate-binding module (CBM) connected through the glycosylated linker peptide. A tunnel-shaped active site rests in the CD and contains 10 glucose unit binding sites. The active site of TrCel7A is lined with four Trp residues with two of them, Trp-40 and Trp-38, in the substrate binding sites near the tunnel entrance. Although addressed in numerous studies the elucidation of the role of CBM and active site aromatics has been obscured by a complex multistep mechanism of processive GHs. Here we studied the role of the CBM-linker and Trp-38 of TrCel7A with respect to binding affinity, on- and off-rates, processivity, and synergism with endoglucanase. The CBM-linker increased the on-rate and substrate affinity of the enzyme. The Trp-38 to Ala substitution resulted in increased off-rates and decreased processivity. The effect of the Trp-38 to Ala substitution on on-rates was strongly dependent on the presence of the CBM-linker. This compensation between CBM-linker and Trp-38 indicates synergism between CBM-linker and CD in feeding the cellulose chain into the active site. The inter-domain synergism was pre-requisite for the efficient degradation of cellulose in the presence of endoglucanase.

Mechanism of product inhibition for cellobiohydrolase Cel7A during hydrolysis of insoluble cellulose

The cellobiohydrolase cellulase Cel7A is extensively utilized in industrial treatment of lignocellulosic biomass under conditions of high product concentrations, and better understanding of inhibition mechanisms appears central in attempts to improve the efficiency of this process. We have implemented an electrochemical biosensor assay for product inhibition studies of cellulases acting on their natural substrate, cellulose. Using this method we measured the hydrolytic rate of Cel7A as a function of both product (inhibitor) concentration and substrate load. This data enabled analyses along the lines of conventional enzyme kinetic theory. We found that the product cellobiose lowered the maximal rate without affecting the Michaelis constant, and this kinetic pattern could be rationalized by two fundamentally distinct molecular mechanisms. One was simple reversibility, that is, an increasing rate of the reverse reaction, lowering the net hydrolytic velocity as product concentrations increase. Strictly this is not a case of inhibition, as no catalytically inactive is formed. The other mechanism that matched the kinetic data was noncompetitive inhibition with an inhibition constant of 490 ± 40 μM. Noncompetitive inhibition implies that the inhibitor binds with comparable strength to either free enzyme or an enzymesubstrate complex, that is, that association between enzyme and substrate has no effect on the binding of the inhibitor. This mechanism is rarely observed, but we argue, that the special architecture of Cel7A with numerous subsites for binding of both substrate and product could give rise to a true noncompetitive inhibition mechanism. Biotechnol. Bioeng. 2016;113: 1178–1186.

Loop variants of the thermophile Rasamsonia emersonii Cel7A with improved activity against cellulose.

Cel7A cellobiohydrolases perform processive hydrolysis on one strand of cellulose, which is threaded through the enzymes substrate binding tunnel. The tunnel structure results from a groove in the catalytic domain, which is covered by a number of loops. These loops have been identified as potential targets for engineering of this industrially important enzyme family, but only few systematic studies on this have been made. Here we show that two asparagine residues (N194 and N197) positioned in the loop covering the glucopyranose subsite −4 (recently denoted B2 loop) of the thermostable Cel7A from Rasamsonia emersonii had profound effects on both substrate interactions and catalytic efficacy. At room temperature the double mutant N194A/N197A showed strongly reduced substrate affinity with a water‐cellulose partitioning coefficient threefold lower than the wild type. Yet, this variant was catalytically efficient with a maximal turnover about twice as high as the wild type. Analogous but smaller changes were found for the single mutants. Analysis of these changes in affinity and kinetics as a function of temperature, led to the conclusion that replacement of N194 and particularly N197 with alanine leads to faster enzyme‐substrate dissociation. Conversely, these residues appeared to have little or no effect on the rate of association. We suggest that the controlled adjustment of the enzyme‐substrate dissociation prompts faster cellulolytic enzymes. Biotechnol. Bioeng. 2017;114: 53–62.

Exo-exo synergy between Cel6A and Cel7A from Hypocrea jecorina: Role of carbohydrate binding module and the endo-lytic character of the enzymes

Synergy between cellulolytic enzymes is essential in both natural and industrial breakdown of biomass. In addition to synergy between endo‐ and exo‐lytic enzymes, a lesser known but equally conspicuous synergy occurs among exo‐acting, processive cellobiohydrolases (CBHs) such as Cel7A and Cel6A from Hypocrea jecorina. We studied this system using microcrystalline cellulose as substrate and found a degree of synergy between 1.3 and 2.2 depending on the experimental conditions. Synergy between enzyme variants without the carbohydrate binding module (CBM) and its linker was strongly reduced compared to the wild types. One plausible interpretation of this is that exo‐exo synergy depends on the targeting role of the CBM. Many earlier works have proposed that exo‐exo synergy was caused by an auxiliary endo‐lytic activity of Cel6A. However, biochemical data from different assays suggested that the endo‐lytic activity of both Cel6A and Cel7A were 103–104 times lower than the common endoglucanase, Cel7B, from the same organism. Moreover, the endo‐lytic activity of Cel7A was 2–3‐fold higher than for Cel6A, and we suggest that endo‐like activity of Cel6A cannot be the main cause for the observed synergy. Rather, we suggest the exo‐exo synergy found here depends on different specificities of the enzymes possibly governed by their CBMs. Biotechnol. Bioeng. 2017;114: 1639–1647.

Direct kinetic comparison of the two cellobiohydrolases Cel6A and Cel7A from Hypocrea jecorina

Cellulose degrading fungi such as Hypocrea jecorina secrete several cellulases including the two cellobiohydrolases (CBHs) Cel6A and Cel7A. The two CBHs differ in catalytic mechanism, attack different ends, belong to different families, but are both processive multi-domain enzymes that are essential in the hydrolysis of cellulose. Here we present a direct kinetic comparison of these two enzymes acting on insoluble cellulose. We used both continuous- and end-point assays under either enzyme- or substrate excess, and found distinct kinetic differences between the two CBHs. Cel6A was catalytically superior with a maximal rate over four times higher than Cel7A. Conversely, the ability of Cel6A to attack diverse structures on the cellulose surface was inferior to Cel7A. This latter difference was pronounced as the density of attack sites for Cel7A was almost an order of magnitude higher compared to Cel6A. We conclude that Cel6A is a fast but selective enzyme and that Cel7A is slower, but promiscuous. One consequence of this is that Cel6A is more effective when substrate is plentiful, while Cel7A excels when substrate is limiting. These diverse kinetic properties of Cel6A and Cel7A might elucidate why both cellobiohydrolases are prominent in cellulolytic degrading fungi.

Thermoactivation of a cellobiohydrolase

We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a “high affinity” variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two‐fold faster around 70 °C. This better thermoactivation of the high‐affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability.

Michaelis - Menten equation for degradation of insoluble substrate

Kinetic studies of homogeneous enzyme reactions where both the substrate and enzyme are soluble have been well described by the Michaelis-Menten (MM) equation for more than a century. However, many reactions are taking place at the interface of a solid substrate and enzyme in solution. Such heterogeneous reactions are abundant both in vivo and in industrial application of enzymes but it is not clear whether traditional enzyme kinetic theory developed for homogeneous catalysis can be applied. Since the molar concentration of surface accessible sites (attack-sites) often is unknown for a solid substrate it is difficult to assess whether the requirement of the MM equation is met. In this paper we study a simple kinetic model, where removal of attack sites expose new ones which preserve the total accessible substrate, and denote this approach the substrate conserving model. The kinetic equations are solved in closed form, both steady states and progress curves, for any admissible values of initial conditions and rate constants. The model is shown to merge with the MM equation and the reverse MM equation when these are valid. The relation between available molar concentration of attack sites and mass load of substrate is analyzed and this introduces an extra parameter to the equations. Various experimental setups to practically and reliably estimate all parameters are discussed.

Anomeric Selectivity and Product Profile of a Processive Cellulase

Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for β-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.