Individual amino acids in the N‐terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases

Abstract

Thermostability and unfolding behavior of the wild-type (1,3-1,4)-P-glucanases from Bacillus macerans (MAC) and Bacillus amyloliquefaciens (AMY) and of two hybrid enzymes H(A12-M)AF14 and H(A12-M)AY13F14A were studied by spectroscopic and microcalorimetric measurements. H(A12-M)AF14 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)AY13F14A, the N-terminal region of MAC is exchanged against the AMY sequence, Y 13 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, are higher in the presence of Ca2+ than in Ca2+ free buffer. Furthermore, the unfolding mechanism is influenced by Ca2+. In Ca2+-free buffer, MAC, H(A12-M)AF14 and H(A12-M)AY13F14A 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)AF14, a two-step unfolding is induced by Ca2+. These data indicate a two-domain structure of AMY and H(A12-M)AF14 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+.

Biochemical and structural characterization of a β-1,3–1,4-glucanase from Bacillus subtilis 168

“…The values of G H 2 O show an intermediate stability of the B. subtilis ␤-1,3-1,4-glucanase compared to the enzymes from Bacillus amyloliquefaciens (5 kcal/mol) and B. macerans (13 kcal/mol) [29]. A hybrid glucanase H(A16-M), in which the 16 N-terminal amino acid of the B. macerans glucanase was replaced by corresponding region of B. amyloliquefaciens enzyme, shows the higher stability with a G H 2 O of 14 and 15 kcal/mol, when complexed with Na + and Ca 2+ respectively [29], confirming that the N-terminal loop in bacterial ␤-1,3-1,4-glucanases contributes to the structural determinants of thermostability [33]. Comparison of the ␤-1,3-1,4-glucanase amino acid sequences from B. subtilis (Genbank No.…”

Myxococcus xanthus spore coat protein S, a stress‐induced member of the βγ‐crystallin superfamily, gains stability from binding of calcium ions

“…Stabilisation of proteins by Ca 2ϩ binding has been frequently observed, e.g. in bovine trypsinogen [32] or bacterial glucanases [33]. The question whether Ca 2ϩ -free protein S resembles a native-like intermediate similar to the one reported for A-lactalbumin [34] needs further investigation.…”

Calcium Binding by the N-Terminal Cellulose-Binding Domain from Cellulomonas fimi β-1,4-Glucanase CenC

“…For example, all have a bulge in the β-sheet containing Asp207 in the 1,3-1,4-β-glucanases and Asp142 in CBD N1 . Also, CBD N1 and the hybrid Bacillus 1,3-1,4β-glucanase H(A16-M) have comparable affinity constants for calcium (18), and are stabilized thermodynamically by the binding of this metal ion (18,(20)(21)(22).…”