title: Atomic resolution structure of a lysine-specific endoproteinase from Lysobacter enzymogenes suggests a hydroxyl group bound to the oxyanion hole.
abstract: Lysobacter enzymogenes lysyl endoproteinase (LysC) is a trypsin-type serine protease with a high pH optimum that hydrolyses all Lys-Xaa peptide bonds. The high specificity of LysC renders it useful for biotechnological purposes. The K30R variant of a related lysyl endoproteinase from Achromobacter lyticus has favourable enzymatic properties that might be transferrable to LysC. To visualize structural differences in the substrate-binding sites, the crystal structures of wild-type and the K30R variant of LysC were determined. The mutation is located at a distance of 12 Å from the catalytic triad and subtly changes the surface properties of the substrate-binding site. The high pH optimum of LysC can be attributed to electrostatic effects of an aromatic Tyr/His stack on the catalytic aspartate and is a general feature of this enzyme subfamily. LysC crystals in complex with the covalent inhibitor N(α)-p-tosyl-lysyl chloromethylketone yielded data to 1.1 and 0.9 Å resolution, resulting in unprecedented precision of the active and substrate-binding sites for this enzyme subfamily. Error estimates on bond lengths and difference electron density indicate that instead of the expected oxyanion a hydroxyl group binds to the partially solvent-exposed oxyanion hole. Protonation of the alkoxide catalytic intermediate might be a recurring feature during serine protease catalysis.
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Asztalos
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Müller
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Werner
authors.initials: W
authors.lastname: Hölke
authors.affiliation: Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
authors.forename: Harald
authors.initials: H
authors.lastname: Sobek
authors.affiliation: Molecular Design and Chemical Biology, F. Hoffmann-La Roche, Grenzacher Strasse 124, 4070 Basel, Switzerland.
authors.forename: Markus G
authors.initials: MG
authors.lastname: Rudolph
mesh_terms: D000838:Anions; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017665:Hydroxyl Radical / Q000378:metabolism*; D008239:Lysine / Q000378:metabolism*; D053528:Lysobacter / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000838:Anions; D017665:Hydroxyl Radical; D010447:Peptide Hydrolases; D008239:Lysine
keywords: Lysobacter enzymogenes; TLCK; catalytic intermediate; chloromethylketone; enzymatic mechanism; lysine-specific endoproteinase; oxyanion hole; substrate specificity