The Mechanism of Cellulose Hydrolysis by a Two-Step, Retaining Cellobiohydrolase Elucidated by Structural and Transition Path Sampling Studies

Abstract

Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.

Kinetics of Cellobiohydrolase (Cel7A) Variants with Lowered Substrate Affinity

“…This is over an order of magnitude less than k cat for the same system (4.8 s Ϫ1 ) (24), and we will therefore not consider this parameter further in the discussion of rate limitation. We note, however, that the above arguments pertain to hydrolysis by monocomponent TrCel7A, and different conclusions regarding k cat may be reached for mixtures of synergistically acting cellulases (21,55). Hence, Jalak et al (21) found that hydrolysis of bacterial cellulose by an optimally composed mixture of Cel7A and the endo-active enzyme Cel5A could reach a specific hydrolysis rate at steady state (about 2 s Ϫ1 ), which was close to the rate constant for the inner catalytic cycle (including hydrolysis, product expulsion, and processive movement).…”

“…We chose Trp-38 as target for a low affinity mutant because this residue has previously been shown to interact with the cellulose chain (8,26). Moreover, it is located in the middle of the tunnel quite far from the scissile bond unlike, for example, Trp-367 and Trp-376, which are directly involved in the twist of the cellulose strand that is important for the actual catalysis (55).…”

“…As outlined in the Introduction, this focus was motivated partly by recent discussions of rate limitation, which have highlighted these two steps, and partly by the deduction that the hydrolytic step (governed by k cat in Scheme 1) is an unlikely candidate for rate determination. The latter relies on numerous measurements of k cat for TrCel7A, which have shown that at room temperature this parameter is in the range of 2-10 s Ϫ1 (19,21,22,24,43,55,60,61), and this is much faster than typically measured specific rates. Because the overall rate is obviously dictated by the slowest step, this discrepancy strongly suggests that catalysis is not the bottleneck and that rate determination must be some much slower process.…”

Genomics Review of Holocellulose Deconstruction by Aspergilli

“…The cellulosic substrate chain has to travel through the tunnel, where ␤-1,4-glycosyl bonds of cellobiose molecules (dimers) are hydrolyzed off the ends (GH6 or GH7 enzymes). The three-dimensional structures are for Trichoderma reesei GH6 (CBHII; PDB entry 1QK2) (108) and GH7 (CBHI or Cel7A; PDB entry 4C4C) enzymes (227).…”

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life

“…The structures of GH7 catalytic domains are known from 10 CBHs, Trichoderma reesei Cel7A (TreCel7A) (12)(13)(14), Heterobasidion irregulare Cel7A (HirCel7A) (15), Phanerochaete chrysosporium Cel7D (PchCel7D) (16), Talaromyces emersonii Cel7A (RemCel7A) (17), Trichoderma harzianum Cel7A (ThaCel7A) (18), Melanocarpus albomyces Cel7B (MalCel7B) (19), Aspergillus fumigatus Cel7A (AfuCel7A) (20), Humicola grisea var. thermoidea Cel7A (HgtCel7A) (21) Limnoria quadripunctata Cel7B (LquCel7B) (22), and Geotrichum candidum Cel7A (23), and three endoglucanases (EGs), T. reesei Cel7B (24), Humicola insolens Cel7B (25), and Fusarium oxysporum Cel7B (26).…”