Identification of novel potential ß-N-acetyl-D-hexosaminidase inhibitors by virtual screening, molecular dynamics simulation and MM-PBSA calculations.

Chitinolytic ß-N-acetyl-d-hexosaminidases, as a class of chitin hydrolysis enzyme in insects, are a potential species-specific target for developing environmentally-friendly pesticides. Until now, pesticides targeting chitinolytic ß-N-acetyl-d-hexosaminidase have not been developed. This study demonstrates a combination of different theoretical methods for investigating the key structural features of this enzyme responsible for pesticide inhibition, thus allowing for the discovery of novel small molecule inhibitors. Firstly, based on the currently reported crystal structure of this protein (OfHex1.pdb), we conducted a pre-screening of a drug-like compound database with 8 × 10(6) compounds by using the expanded pesticide-likeness criteria, followed by docking-based screening, obtaining 5 top-ranked compounds with favorable docking conformation into OfHex1. Secondly, molecular docking and molecular dynamics simulations are performed for the five complexes and demonstrate that one main hydrophobic pocket formed by residues Trp424, Trp448 and Trp524, which is significant for stabilization of the ligand-receptor complex, and key residues Asp477 and Trp490, are respectively responsible for forming hydrogen-bonding and π-π stacking interactions with the ligands. Finally, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis indicates that van der Waals interactions are the main driving force for the inhibitor binding that agrees with the fact that the binding pocket of OfHex1 is mainly composed of hydrophobic residues. These results suggest that screening the ZINC database can maximize the identification of potential OfHex1 inhibitors and the computational protocol will be valuable for screening potential inhibitors of the binding mode, which is useful for the future rational design of novel, potent OfHex1-specific pesticides.

Mutational effects on transglycosylating activity of family 18 chitinases and construction of a hypertransglycosylating mutant.

Enzymatic features that determine transglycosylating activity have been investigated through site-directed mutagenesis studies on two family 18 chitinases, ChiA and ChiB from Serratia marcescens, with inherently little transglycosylation activity. The activity was monitored for the natural substrate (GlcNAc)(4) using mass spectrometry and HPLC. Mutation of the middle Asp in the diagnostic DxDxE motif, which interacts with the catalytic Glu during the catalytic cycle, yielded the strongly transglycosylating mutants ChiA-D313N and ChiB-D142N, respectively. Mutation of the same Asp(313/142) to Ala or the mutation of Asp(311/140) to either Asn or Ala had no or much smaller effects on transglycosylating activity. Mutation of Phe(396) in the +2 subsite of ChiA-D313N to Trp led to a severalfold increase in transglycosylation rate while replacement of aromatic residues with Ala in the aglycon (sugar acceptor-binding) subsites of ChiA-D313N and ChiB-D142N led to a clear reduction in transglycosylating activity. Taken together, these results show that the transglycosylation properties of family 18 chitinases may be manipulated by mutations that affect the configuration of the catalytic machinery and the affinity for sugar acceptors. The hypertransglycosylating mutant ChiA-D313N-F396W may find applications for synthetic purposes.

Structure of Saccharomyces cerevisiae chitinase 1 and screening-based discovery of potent inhibitors.

Chitinases hydrolyse the beta(1,4)-glycosidic bonds of chitin, an essential fungal cell wall component. Genetic data on a subclass of fungal family 18 chitinases have suggested a role in cell wall morphology. Specific inhibitors of these enzymes would be useful as tools to study their role in cell wall morphogenesis and could possess antifungal properties. Here, we describe the crystallographic structure of a fungal "plant-type" family 18 chitinase, that of Saccharomyces cerevisiae CTS1. The enzyme is active against 4-methylumbelliferyl chitooligosaccharides and displays an unusually low pH optimum for activity. A library screen against ScCTS1 yielded hits with Ki 's as low as 3.2 microM. Crystal structures of ScCTS1 in complex with inhibitors from three series reveal striking mimicry of carbohydrate substrate by small aromatic moieties and a pocket that could be further exploited in optimization of these inhibitors.