Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase.


Abstract

Chloride-dependent alpha-amylases constitute a well conserved family of enzymes thereby allowing investigation of the characteristics of each member to understand, for example, relevant properties required for environmental adaptation. In this context, we have constructed a double mutant (Q58C/A99C) of the cold-active and heat-labile alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis, defined on the basis of its strong similarity with the mesophilic enzyme from pig pancreas. This mutant was characterized to understand the role of an extra disulfide bond specific to warm-blooded animals and located near the entrance of the catalytic cleft. We show that the catalytic parameters of the mutant are drastically modified and similar to those of the mesophilic enzyme. Calorimetric studies demonstrated that the mutant is globally stabilized (DeltaDeltaG = 1.87 kcal/mol at 20 degrees C) when compared with the wild-type enzyme, although the melting point (T(m)) was not increased. Moreover, fluorescence quenching experiments indicate a more compact structure for the mutated alpha-amylase. However, the strain imposed on the active site architecture induces a 2-fold higher thermal inactivation rate at 45 degrees C as well as the appearance of a less stable calorimetric domain. It is concluded that stabilization by the extra disulfide bond arises from an enthalpy-entropy compensation effect favoring the enthalpic contribution. Study holds ProTherm entries: 17640, 17641, 17642, 17643, 17644, 17645, 17646, 17647, 17648 Extra Details: Chloride-dependent; cold-active; catalytic parameters; compact structure;,enthalpy-entropy compensation effect

Submission Details

ID: CTSS9to63

Submitter: Connie Wang

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

Version: 1

Publication Details
D'Amico S;Gerday C;Feller G,J. Biol. Chem. (2002) Dual effects of an extra disulfide bond on the activity and stability of a cold-adapted alpha-amylase. PMID:12324460
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
1AQH 1997-07-30T00:00:00+0000 2.0 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1AQM 1997-07-31T00:00:00+0000 1.85 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS COMPLEXED WITH TRIS
1B0I 1998-11-10T00:00:00+0000 2.4 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1G94 2000-11-22T00:00:00+0000 1.74 CRYSTAL STRUCTURE ANALYSIS OF THE TERNARY COMPLEX BETWEEN PSYCHROPHILIC ALPHA AMYLASE FROM PSEUDOALTEROMONAS HALOPLANCTIS IN COMPLEX WITH A HEPTA-SACCHARIDE AND A TRIS MOLECULE
1G9H 2000-11-23T00:00:00+0000 1.8 TERNARY COMPLEX BETWEEN PSYCHROPHILIC ALPHA-AMYLASE, COMII (PSEUDO TRI-SACCHARIDE FROM BAYER) AND TRIS (2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL)
1JD7 2001-06-13T00:00:00+0000 2.25 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300R OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1JD9 2001-06-13T00:00:00+0000 2.5 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1KXH 2002-01-31T00:00:00+0000 2.3 Crystal structure of the complex between an inactive mutant of psychrophilic alpha-amylase (D174N) and acarbose
1L0P 2002-02-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE ANALYSIS OF THE COMPLEX BETWEEN PSYCHROPHILIC ALPHA AMYLASE FROM PSEUDOALTEROMONAS HALOPLANCTIS AND NITRATE
1BVN 1998-09-16T00:00:00+0000 2.5 PIG PANCREATIC ALPHA-AMYLASE IN COMPLEX WITH THE PROTEINACEOUS INHIBITOR TENDAMISTAT

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Alpha-amylase P29957 AMY_PSEHA
98.4 Pancreatic alpha-amylase P00690 AMYP_PIG