Distal Hydrophobic Loop Modulates the Copper Active Site and Reaction of AA13 Polysaccharide Monooxygenases.

Polysaccharide monooxygenases (PMOs) use a type-2 copper center to activate O2 for the selective hydroxylation of one of the two C-H bonds of glycosidic linkages. Our electron paramagnetic resonance (EPR) analysis and molecular dynamics (MD) simulations suggest the unprecedented dynamic roles of the loop containing the residue G89 (G89 loop) on the active site structure and reaction cycle of starch-active PMOs (AA13 PMOs). In the Cu(II) state, the G89 loop could switch between an "open" and "closed" conformation, which is associated with the binding and dissociation of an aqueous ligand in the distal site, respectively. The conformation of the G89 loop influences the positioning of the copper center on the preferred substrate of AA13 PMOs. The dissociation of the distal ligand results in the bending of the T-shaped core of the Cu(II) active site, which could help facilitate its reduction to the active Cu(I) state. In the Cu(I) state, the G89 loop is in the "closed" conformation with a confined copper center, which could allow for efficient O2 binding. In addition, the G89 loop remains in the "closed" conformation in the Cu(II)-superoxo intermediate, which could prevent off-pathway superoxide release via exchange with the distal aqueous ligand. Finally, at the end of the reaction cycle, aqueous ligand binding to the distal site could switch the G89 loop to the "open" conformation and facilitate product release.

Interaction of carbohydrate binding module 20 with starch substrates.

CBM20s are starch-binding domains found in many amylolytic enzymes, including glucoamylase, alpha-amylase, beta-amylases, and a new family of starch-active polysaccharide monooxygenases (AA13 PMOs). Previous studies of CBM20-substrate interaction only concerned relatively small or soluble amylose molecules, while amylolytic enzymes often work on extended chains of insoluble starch molecules. In this study, we utilized molecular simulation techniques to gain further insights into the interaction of CBM20 with substrates of various sizes via its two separate binding sites, termed as BdS1 and BdS2. Results show that substrate binding at BdS1 involving two conserved tryptophan residues is about 2-4 kcal mol-1 stronger than that at BdS2. CBM20 exhibits about two-fold higher affinity for helical substrates than for the amylose random coils. The affinity for amylose individual double helices does not depend on the helices' length. At least three parallel double helices are required for optimal binding. The binding affinity for a substrate containing 3 or more double helices is ∼-15 kcal mol-1, which is 2-3 kcal mol-1 larger than that for individual double helices. 100 ns molecular dynamics simulations were carried out for the binding of CBM20 to an extended substrate containing 3 layers of 9 60-unit double helices (A3L). A stable conformation of CBM20-A3L was found at BdS1. However, when CBM20 binds A3L viaBdS2, it moves across the surface of the substrate and does not form a stable complex. MD simulations show that small amylose helices are quickly disrupted upon binding to CBM20. Our results provide some important molecular insights into the interactions of CBM20 with starch substrates, which will serve as the basis for further studies of CBM20-containing enzymes, including AA13 PMOs.