A Water-Assisted Catalytic Mechanism in Glycoside Hydrolases Demonstrated on the Staphylococcus aureus Autolysin E

Abstract

Autolysin E (AtlE), from Staphylococcus aureus, is a cell-wall-degrading enzyme that is a potential drug target. It is a member of the glycoside hydrolase (GH) class, enzymes that commonly have either two catalytic residues and hydrolyze their substrates by inverting or retaining mechanisms or one catalytic residue and undergo retaining, substrate-assisted catalysis. Here, we address the catalytic mechanism of AtlE. Site-directed mutagenesis studies identified Glu138 as the only catalytic residue. Quantum mechanics/molecular mechanics (QM/MM) simulations of the possible reaction pathways suggest that hydrolysis proceeds via a retaining, water-assisted mechanism and an oxocarbenium ion like transition state. These results, on the basis of data from a member of the hydrolase GH73 family, support the hypothesis of the presence of an alternative catalytic mechanism in glycoside hydrolases, which can be considered in the design of future AtlE inhibitors.

Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

“…The mutation of the tyrosine residue Y214 in the YATD region to alanine severely affected enzyme activity, whereas mutation of Y214 to phenylalanine completely restored the substrate degrading ability. Similar observations were previously established for AtlE 17 , as well as for four other GH73 enzymes 24,25,28,32,33 , demonstrating that an aromatic residue is required at this position. The T216A and D217N mutants exhibited a marked decrease in activity.…”

“…The T216A and D217N mutants exhibited a marked decrease in activity. Similar observations were made for the equivalent S226A and D227A mutants in AtlE 17 . We were unable to obtain a soluble D217A mutant from AtlA-gl, indicating that D217 is important for the structural stability of the region and protein folding.…”

“…To identify the equivalent residues in AtlA-gl involved in substrate binding as in AtlE 17 , we introduced point mutations that either targeted the catalytic Glu directly or the substrate binding YA(S/T)D region (Fig. 4).…”

“…We recently determined the crystal structures of a homologous protein, autolysin E (AtlE) in complex with substrate fragments, which we used together with other available structural data on lysozymes to build a model of (NAG-NAM) 3 substrate binding to AtlE 16 . We have also analyzed the amino acids that are important for the reaction to transpire and identified E138 as the catalytic general acid and Y224-A225-S226-D227 as the motif responsible for substrate binding 17 .…”

“…AtlA has been shown to be localized at the septal region and to be involved in cell division 14,21 . We combined the understanding of substrate binding obtained from our previous AtlE studies 16,17 with insights obtained from the SagB and AtlA-gl structures. The analysis of substrate binding sites revealed that for the active site residues to enable the enzymatic reaction to proceed, a substantial conformational change has to take place.…”