title: Expression, purification, characterization, and X-ray analysis of selenomethionine 215 variant of leukocyte collagenase.
abstract: Matrix metalloproteinases belong to the superfamily of metzincins containing, besides a similar topology and a strictly conserved zinc environment, a 1,4-tight turn with a strictly conserved methionine residue at position three (the so called Met-turn [Bode et. al. (1993) FEBS 331, 134-140; Stöcker et al. (1995) Protein Sci. 4, 823-840]. The distal S-CH3 moiety of this methionine residue forms the hydrophobic basement of the three His residues liganding the catalytic zinc ion. To assess the importance of this methionine, we have expressed the catalytic domain of neutrophil collagenase (rHNC, residues Met80-Gly242) in the methionine auxotrophic Escherichia coli strain B834[DE3](hsd metB), with the two methionine residues replaced by selenomethionine. Complete replacement was confirmed by amino acid analysis and electrospray mass spectrometry. The folded and purified enzyme retained its catalytic activity, but showed modifications which are reflected in change kinetic parameters. The Met215SeMet substitution caused a decrease in conformational stability upon area denaturation. The X-ray crystal structure of this selenomethionine rHNC was virtually identical to that of the wild-type catalytic domain except for a very faint local disturbance around the sulfur-seleno substitution site.
authors.affiliation: Fakultät für Chemie und Biochemie, Universität Bielefeld, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Pieper
authors.forename: M
authors.initials: M
authors.lastname: Betz
authors.forename: N
authors.initials: N
authors.lastname: Budisa
authors.forename: F X
authors.initials: FX
authors.lastname: Gomis-Rüth
authors.forename: W
authors.initials: W
authors.lastname: Bode
authors.forename: H
authors.initials: H
authors.lastname: Tschesche
mesh_terms: D003001:Cloning, Molecular; D017364:Collagenases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018360:Crystallography, X-Ray; D007962:Leukocytes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D012645:Selenomethionine / Q000737:chemistry*; D013057:Spectrum Analysis
chemical_list: D012645:Selenomethionine; D017364:Collagenases