Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes.


Abstract

In a comparative investigation on two thermostable alpha-amylases [Bacillus amyloliquefaciens (BAA), T(m) = 86 degrees C and Bacillus licheniformis (BLA), T(m) = 101 degrees C], we studied thermal and guanidine hydrochloride (GndHCl)-induced unfolding using fluorescence and CD spectroscopy, as well as dynamic light scattering. Depletion of calcium from specific ion-binding sites in the protein structures reduces the melting temperature tremendously for both alpha-amylases. The reduction is nearly the same for both enzymes, namely, in the order of 50 degrees C. Thus, the difference in thermostability between BLA and BAA (DeltaT(m) approximately 15 degrees C) is related to intrinsic properties of the respective protein structures themselves and is not related to the strength of ion binding. The thermal unfolding of both proteins is characterized by a full disappearance of secondary structure elements and by a concurrent expansion of the 3D structure. GndHCl-induced unfolding also yields a fully vanishing secondary structure but with more expanded 3D structures. Both alpha-amylases remain much more compact upon thermal unfolding as compared to the fully unfolded state induced by chemical denaturants. Such rather compact thermal unfolded structures lower the conformational entropy change during the unfolding transition, which principally can contribute to an increased thermal stability. Structural flexibilities of both enzymes, as measured with tryptophan fluorescence quenching, are almost identical for both enzymes in the native states, as well as in the unfolded states. Furthermore, we do not observe any difference in the temperature dependence of the structural flexibilities between BLA and BAA. These results indicate that conformational dynamics on the time scale of our studies seem not to be related to thermal stability or to thermal adaptation. Study holds ProTherm entries: 17214, 17215, 17216, 17217, 17218, 17219, 17220, 17221 Extra Details: thermostable; calcium; ion-binding sites; conformational entropy; structural flexibilities

Submission Details

ID: cPhCybVZ

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Fitter J;Haber-Pohlmeier S,Biochemistry (2004) Structural stability and unfolding properties of thermostable bacterial alpha-amylases: a comparative study of homologous enzymes. PMID:15274613
Additional Information

Study Summary

Number of data points 12
Proteins GLYCOSYLTRANSFERASE ; Alpha-amylase ; Alpha-amylase
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: Tm buffers:Mops: 10 mM, details:Additives EDTA (5 mM),, ionic:NaCl: 50 mM, prot_conc:0.13 mg/mL ; Experimental Assay: dHvH details:Additives EDTA (5 mM),, ionic:NaCl: 50 mM ; Experimental Assay: Tm ionic:NaCl,CaCl2: 50 mM,2mM, details:Additives , buffers:Mops: 10 mM, prot_conc:0.13 mg/mL ; Experimental Assay: dHvH details:Additives , ionic:NaCl,CaCl2: 50 mM,2mM ; Experimental Assay: Tm prot_conc:0.05 mg/mL, buffers:Mops: 30 mM, details:Additives EDTA (5 mM),, ionic:NaCl: 50 mM ; Experimental Assay: Tm prot_conc:0.05 mg/mL, buffers:Mops: 30 mM, ionic:NaCl,CaCl2: 50 mM,2mM, details:Additives
Libraries Mutations for sequence A:ANLNGTLMQYFEWYMPNDGQHWKRLQNDSAYLAEHGITAVWIPPAYKGTSQADVGYGAYDLYDLGEFHQKGTVRTKYGTKGELQSAIKSLHSRDINVYGDVVINHKGGADATEDVTAVEVDPADRNRVISGEHLIKAWTHFHFPGRGSTYSDFKWHWYHFDGTDWDESRKLNRIYKFQGKAWDWEVSNE/B:NGNYDYLMYADIDYDHPDVAAEIKRWGTWYANELQLDGFRLDAVKHIKFSFLRDWVNHVREKTGKEMFTVAEYWQNDLGALENYLNKTNFNHSVFDVPLHYQFHAASTQGGGYDMRKLLNSTVVSKHPLKAVTFVDNHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGDSQREIPALKHKIEPILKARKQYAYGAQHDYFDHHDIVGWTREGDSSVANSGLAALITDGPGGAKRMYVGRQNAGETWHDITGNRSEPVVINSEGWGEFHVNGGSVSIYVQR ; Mutations for sequence MIQKRKRTVSFRLVLMCTLLFVSLPITKTSAVNGTLMQYFEWYTPNDGQHWKRLQNDAEHLSDIGITAVWIPPAYKGLSQSDNGYGPYDLYDLGEFQQKGTVRTKYGTKSELQDAIGSLHSRNVQVYGDVVLNHKAGADATEDVTAVEVNPANRNQETSEEYQIKAWTDFRFPGRGNTYSDFKWHWYHFDGADWDESRKISRIFKFRGEGKAWDWEVSSENGNYDYLMYADVDYDHPDVVAETKKWGIWYANELSLDGFRIDAAKHIKFSFLRDWVQAVRQATGKEMFTVAEYWQNNAGKLENYLNKTSFNQSVFDVPLHFNLQAASSQGGGYDMRRLLDGTVVSRHPEKAVTFVENHDTQPGQSLESTVQTWFKPLAYAFILTRESGYPQVFYGDMYGTKGTSPKEIPSLKDNIEPILKARKEYAYGPQHDYIDHPDVIGWTREGDSSAAKSGLAALITDGPGGSKRMYAGLKNAGETWYDITGNRSDTVKIGSDGWGEFHVNDGSVSIYVQK

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
3BH4 2008-12-09 1.4 High resolution crystal structure of Bacillus amyloliquefaciens alpha-amylase
1VJS 1997-03-12 1.7 STRUCTURE OF ALPHA-AMYLASE PRECURSOR
1E43 2001-06-21 1.7 Native structure of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 1.7A
1OB0 2003-01-30 1.83 Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface
1E3X 2001-06-21 1.9 Native structure of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 1.92A
1BLI 1999-03-23 1.9 BACILLUS LICHENIFORMIS ALPHA-AMYLASE
1E3Z 2001-06-21 1.93 Acarbose complex of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 1.93A
1BPL 1996-08-17 2.2 GLYCOSYLTRANSFERASE
1E40 2001-06-21 2.2 Tris/maltotriose complex of chimaeric amylase from B. amyloliquefaciens and B. licheniformis at 2.2A

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
197.4 A,B GLYCOSYLTRANSFERASE P06278 AMY_BACLI
100.0 Alpha-amylase P00692 AMY_BACAM