Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library

Abstract

Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.

Heterologous expression of peptidyl-Lys metallopeptidase ofArmillaria melleaand mutagenic analysis of the recombinant peptidase

“…The two structures had a RMSD of 0.001 on backbone residues and 0.239 on the side chain residues (2). In this model Am-LysN has a narrow, and relatively short (57 amino acid) binding cleft.…”

“…The homology model of Am-LysN was built in MOE (2,11). Engineering of the protease was done in the presence of fitted substrates, and new molecular dynamic simulations based on the previously optimized model were run for 1 h at 350 kelvin to accommodate the changes (2).…”

“…Fushimi et al showed that the aspartic acid D132 is necessary to coordinate zinc binding of the homologous protein deuterolysin (8). A 7-amino acid peptide was fitted into the binding cleft of Am-LysN using molecular dynamics (2).We found two aspartic acids and two glutamic acids forming a negatively charged cavity causing the strict lysine specificity. Binding of an arginine's guanidinium group to the E159 of the active site, distorts the peptide backbone away from the zinc, rendering the substrate unsuited for catalysis.…”

“…Peptidyl-Lys metallopeptidase (LysN) from Armillaria mellea is an aspzincin metalloprotease, which cleaves in front of lysines (2). Hence, it is unusual by having its substrate specificity at the C-terminal side of the scissile bond (/K) (3).…”

“…This feature can be used to facilitate de novo peptide mapping. Recently we profiled the substrate specificity of AmLysN and found it to be strictly lysine specific at S 1 0 , and having decreasing specificity when moving away from the scissile bond (2,6).…”