Influence of Ca2+ on conformation and stability of three bacterial hybrid glucanases

The three hybrid glucanases (1–12)AMY · MAC(13–214), (1–12)AMY · des-Tyr13MAC(14–214); (1–16)AMY · 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 · 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 · MAC(17–214) and (1–12)AMY · 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 · MAC(13–214), which differs from (1–12)AMY · 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.