Individual amino acids in the N-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases.
Thermostability and unfolding behavior of the wild-type (1,3-1,4)-beta-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M) delta F14 and H(A12-M) delta Y13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12-M) delta F14 is constructed by the fusion of 12 N-terminal amino acids of AMY with amino acids 13-214 of MAC, and by deletion of F14. In H(A12-M) delta Y13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y13 is deleted, and Phe 14 is exchanged against Ala. The sequence of the N-terminal loop region from Pro 9 to amino acid 16 (or 17) is very important for the properties of the enzymes and influences the effects of Ca2+ ions on the thermostability and unfolding behavior of the enzymes. The half transition temperatures T(m) are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca(2+)-free buffer, MAC, H(A12-M) delta F14 and H(A12-M) delta Y13F14A unfold in a single cooperative transition from the folded state to the unfolded state, whereas for AMY, a two-step unfolding was found. In the presence of Ca2+, the two-step unfolding of AMY is strengthened. Furthermore, for H(A12-M) delta F14, a two-step unfolding is induced by Ca2+. These data indicate a two-domain structure of AMY and H(A12-M) delta F14, in the presence of Ca2+. Thus, point mutations in a peripheral loop region are decisive for thermal stabilities and unfolding mechanisms of the studied glucanases in the presence of Ca2+.
Welfle
K
K
Institute of Biology, Faculty I for Mathematical and Natural Sciences, Humboldt-University, Berlin, Germany. welfle@orion.rz.mdc.berlin.de
Misselwitz
R
R
Politz
O
O
Borriss
R
R
Welfle
H
H
D001407:Bacillus / Q000201:enzymology*; D002118:Calcium / Q000737:chemistry; D002152:Calorimetry, Differential Scanning; D044644:Endo-1,3(4)-beta-Glucanase; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines; D008958:Models, Molecular; D011489:Protein Denaturation; D017510:Protein Folding; D013696:Temperature; D013816:Thermodynamics
D006146:Guanidines; D006026:Glycoside Hydrolases; D044644:Endo-1,3(4)-beta-Glucanase; D019791:Guanidine; D002118:Calcium