Aromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases

Abstract

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to deconstruct recalcitrant polysaccharides to sugars. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface after each hydrolytic step; they are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. The ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts is a notable feature of processive glycoside hydrolases. We hypothesized that these aromatic residues have uniquely defined roles, such as substrate chain acquisition and binding in the catalytic tunnel, that are defined by their local environment and position relative to the substrate and the catalytic center. Here, we investigated this hypothesis with variants of Serratia marcescens family 18 processive chitinases ChiA and ChiB. We applied molecular simulation and free energy calculations to assess active site dynamics and ligand binding free energies. Isothermal titration calorimetry provided further insight into enthalpic and entropic contributions to ligand binding free energy. Thus, the roles of six aromatic residues, Trp-167, Trp-275, and Phe-396 in ChiA, and Trp-97, Trp-220, and Phe-190 in ChiB, have been examined. We observed that point mutation of the tryptophan residues to alanine results in unfavorable changes in the free energy of binding relative to wild-type. The most drastic effects were observed for residues positioned at the "entrances" of the deep substrate-binding clefts and known to be important for processivity. Interestingly, phenylalanine mutations in ChiA and ChiB had little to no effect on chito-oligomer binding, in accordance with the limited effects of their removal on chitinase functionality.

Transglycosylation by a chitinase from Enterobacter cloacae subsp. cloacae generates longer chitin oligosaccharides

“…Single amino acid residue present immediate to the chitin-binding motif and presence of α/β fold insertion plays a pivotal role in processivity 39 . The two aromatic amino acids Trp97 and Trp220 were crucial for processivity in ChiB from S. marcescens 40 . Presumably, corresponding aromatic amino acids Trp114 and Tyr226 might tightly bind with the polymeric substrate and steer the processive action of Ec Chi1, implying that Ec Chi1 might be a processive enzyme (Fig.…”

Multiscale Characterization of Complex Binding Interactions of Cellulolytic Enzymes Highlights Limitations of Classical Approaches

“…Molecular dynamics simulations have been employed extensively to study cellulolytic enzymes (22,(57)(58)(59)(60), and can offer detailed atomistic insights into the highly heterogeneous and multimodal CAZyme binding interactions with insoluble polysaccharides. MD simulations have also revealed structural and dynamical features of cellulose III such as hydrogen bonding patterns, solvent accessible surface area, and decrystallization free energy (17,43).…”

Ega3 from the fungal pathogen Aspergillus fumigatus is an endo-α-1,4-galactosaminidase that disrupts microbial biofilms

“…tunnel-forming tryptophan. Tunnel formation has been strongly correlated with processive activity where once bound, and the polysaccharide substrate may be cleaved repetitively from one end (27,49,53,54). Endo-acting processive enzymes have been found to produce predominantly short oligosaccharides, including di-to tetrasaccharides from polysaccharide substrates.…”