The hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function

Abstract

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall–modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the −1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the −1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

Non-enzymatic action of expansins

“…From their simulations of endoglucanase Cel45A, Bharadwaj et al (1) propose that structurally related expansins and MltA may cut glycan backbones without generating reducing ends. This is tenable for MltA, a peptidoglycan lytic transglycosylase whose action produces nonreducing 1,6-anhydro products, but is untenable for expansins.…”

QM/MM Simulations of Enzymatic Hydrolysis of Cellulose: Probing the Viability of an Endocyclic Mechanism for an Inverting Cellulase

“…This step exhibits a reaction barrier of magnitude comparable to (and even lower than) the steps of exocyclic pathways of GH12 (22 kcal/mol), 66 GH38 (23 kcal/mol), 67 GH43 (24 kcal/mol), 68 GH8 (36 kcal/mol), 69 and GH16 (32 kcal/mol). 70 Recently, Bharadwaj et al 49 reported a similar activation barrier of 23.6 kcal/mol and reaction free energy of approximately 4 kcal/mol for a GH45 subfamily A inverting endoglucanase from Humicola insolens (HiCel45A) using unbiased QM/MM simulations based on the transition path sampling techniques.…”

“…In particular, a very recent investigation on the hydrolysis mechanism of a GH45 subfamily A cellulase reported a similar barrier (23.55 ± 0.3 kcal/mol) using extensive unbiased transition path sampling QM/MM simulations. 49 …”

A novel Swollenin from Talaromyces leycettanus JCM12802 enhances cellulase hydrolysis towards various substrates

“…In general, expansins have a length of no more than 250 amino acids which constitute a twodomain structure. The primary domain resembles the glycoside hydrolase family 45 (GH45) and such homology preserves certain sequence features of the GH45 catalytic site [15]. The second domain has a characteristic at aromatic-rich surface and is homologous to group-2 grass pollen allergens.…”