title: Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases.
abstract: The three hybrid glucanases (1-12)AMY x MAC(13-214), (1-12)AMY x des-Tyr13MAC(14-214); (1-16)AMY x MAC(17-214) are composed of short N-terminal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13-214, 14-214 and 17-214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by the similar characteristics of their CD spectra in the far- and near-ultraviolet region. A metal-ion-binding site was identified in the hybrid glucanase (1-16)AMY x MAC(17-214) by a crystal structure analysis [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287-5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or absence of Ca2+ ions but for (1-16)AMY x MAC(17-214) and (1-12)AMY x des-Tyr13MAC(14-214) the occupation of this metal-binding site by a Ca2+ ion is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1-12)AMY x MAC(13-214), which differs from (1-12)AMY x des-Tyr13MAC(14-214) by only one additional amino acid in an N-terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues near the N-terminus of the B. macerans glucanase against amino acids found at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and concomitantly the stability of the hybrid glucanases.
authors.affiliation: Institute of Biology, Math.-Nat. Faculty I, Humboldt-University, Berlin, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Welfle
authors.forename: R
authors.initials: R
authors.lastname: Misselwitz
authors.forename: H
authors.initials: H
authors.lastname: Welfle
authors.forename: O
authors.initials: O
authors.lastname: Politz
authors.forename: R
authors.initials: R
authors.lastname: Borriss
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D010446:Peptide Fragments; D006026:Glycoside Hydrolases; D002118:Calcium