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)-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+. Study holds ProTherm entries: 7758, 7759, 7760, 7761, 7762, 7763, 7764, 7765, 7766, 7767, 7768, 7769, 7770, 7771, 7772, 7773, 7774, 7775, 7776, 7777, 7778, 7779, 7780 Extra Details: MAC: Bacillus macerans (1,3-1,4)-beta-glucanase unfolding

Submission Details

ID: PTByQWJ33

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Welfle K;Misselwitz R;Politz O;Borriss R;Welfle H,Protein Sci. (1996) Individual amino acids in the N-terminal loop region determine the thermostability and unfolding characteristics of bacterial glucanases. PMID:8931144
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
2AYH 1995-03-31 1.6 CRYSTAL AND MOLECULAR STRUCTURE AT 1.6 ANGSTROMS RESOLUTION OF THE HYBRID BACILLUS ENDO-1,3-1,4-BETA-D-GLUCAN 4-GLUCANOHYDROLASE H(A16-M)
1U0A 2005-09-06 1.64 Crystal structure of the engineered beta-1,3-1,4-endoglucanase H(A16-M) in complex with beta-glucan tetrasaccharide
1AJK 1998-05-06 1.8 CIRCULARLY PERMUTED (1-3,1-4)-BETA-D-GLUCAN 4-GLUCANOHYDROLASE CPA16M-84
1CPN 1994-06-22 1.8 NATIVE-LIKE IN VIVO FOLDING OF A CIRCULARLY PERMUTED JELLYROLL PROTEIN SHOWN BY CRYSTAL STRUCTURE ANALYSIS
1GLH 1995-02-07 2.0 CATION BINDING TO A BACILLUS (1,3-1,4)-BETA-GLUCANASE. GEOMETRY, AFFINITY AND EFFECT ON PROTEIN STABILITY
1CPM 1994-06-22 2.0 NATIVE-LIKE IN VIVO FOLDING OF A CIRCULARLY PERMUTED JELLYROLL PROTEIN SHOWN BY CRYSTAL STRUCTURE ANALYSIS
1AJO 1998-05-06 2.07 CIRCULARLY PERMUTED (1-3,1-4)-BETA-D-GLUCAN 4-GLUCANOHYDROLASE CPA16M-127
1AXK 1999-05-11 2.1 ENGINEERED BACILLUS BIFUNCTIONAL ENZYME GLUXYN-1
3O5S 2011-07-13 2.2 Crystal Structure of the endo-beta-1,3-1,4 glucanase from Bacillus subtilis (strain 168)
1MAC 1995-02-27 2.3 CRYSTAL STRUCTURE AND SITE-DIRECTED MUTAGENESIS OF BACILLUS MACERANS ENDO-1,3-1,4-BETA-GLUCANASE
1BYH 1993-10-31 2.8 MOLECULAR AND ACTIVE-SITE STRUCTURE OF A BACILLUS (1-3,1-4)-BETA-GLUCANASE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.4 Beta-glucanase P04957 GUB_BACSU
100.0 Beta-glucanase P07980 GUB_BACAM
100.0 Beta-glucanase P23904 GUB_PAEMA
94.2 Beta-glucanase P45797 GUB_PAEPO