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
1HX0 2001-08-08 1.38 Structure of pig pancreatic alpha-amylase complexed with the 'truncate' acarbose molecule (pseudotrisaccharide)
1KXV 2002-06-19 1.6 Camelid VHH Domains in Complex with Porcine Pancreatic alpha-Amylase
1KXQ 2002-06-19 1.6 Camelid VHH Domain in Complex with Porcine Pancreatic alpha-Amylase
1G94 2001-12-12 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 2002-06-26 1.8 TERNARY COMPLEX BETWEEN PSYCHROPHILIC ALPHA-AMYLASE, COMII (PSEUDO TRI-SACCHARIDE FROM BAYER) AND TRIS (2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL)
1DHK 1997-12-24 1.85 STRUCTURE OF PORCINE PANCREATIC ALPHA-AMYLASE
1AQM 1999-03-02 1.85 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS COMPLEXED WITH TRIS
3L2M 2010-04-14 1.97 X-ray Crystallographic Analysis of Pig Pancreatic Alpha-Amylase with Alpha-cyclodextrin
1KXT 2002-06-19 2.0 Camelid VHH Domains in Complex with Porcine Pancreatic alpha-Amylase
1AQH 1999-02-16 2.0 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1WO2 2005-03-15 2.01 Crystal structure of the pig pancreatic alpha-amylase complexed with malto-oligosaacharides under the effect of the chloride ion
1UA3 2003-10-14 2.01 Crystal structure of the pig pancreatic a-amylase complexed with malto-oligosaccharides
1JFH 1998-12-02 2.03 STRUCTURE OF A PANCREATIC ALPHA-AMYLASE BOUND TO A SUBSTRATE ANALOGUE AT 2.03 ANGSTROM RESOLUTION
1L0P 2002-06-19 2.1 CRYSTAL STRUCTURE ANALYSIS OF THE COMPLEX BETWEEN PSYCHROPHILIC ALPHA AMYLASE FROM PSEUDOALTEROMONAS HALOPLANCTIS AND NITRATE
3L2L 2010-04-14 2.11 X-ray Crystallographic Analysis of Pig Pancreatic Alpha-Amylase with Limit Dextrin and Oligosaccharide
1PPI 1995-05-24 2.2 THE ACTIVE CENTER OF A MAMMALIAN ALPHA-AMYLASE. THE STRUCTURE OF THE COMPLEX OF A PANCREATIC ALPHA-AMYLASE WITH A CARBOHYDRATE INHIBITOR REFINED TO 2.2 ANGSTROMS RESOLUTION
1PIG 1996-12-07 2.2 PIG PANCREATIC ALPHA-AMYLASE COMPLEXED WITH THE OLIGOSACCHARIDE V-1532
1JD7 2002-09-18 2.25 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300R OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1PIF 1996-12-07 2.3 PIG ALPHA-AMYLASE
1OSE 1997-04-01 2.3 Porcine pancreatic alpha-amylase complexed with acarbose
1KXH 2002-06-19 2.3 Crystal structure of the complex between an inactive mutant of psychrophilic alpha-amylase (D174N) and acarbose
1VAH 2005-04-26 2.4 Crystal structure of the pig pancreatic-amylase complexed with r-nitrophenyl-a-D-maltoside
1B0I 1999-11-17 2.4 ALPHA-AMYLASE FROM ALTEROMONAS HALOPLANCTIS
1JD9 2002-09-18 2.5 CRYSTAL STRUCTURE ANALYSIS OF THE MUTANT K300Q OF PSEUDOALTEROMONAS HALOPLANCTIS ALPHA-AMYLASE
1BVN 1998-09-23 2.5 PIG PANCREATIC ALPHA-AMYLASE IN COMPLEX WITH THE PROTEINACEOUS INHIBITOR TENDAMISTAT
4X0N 2015-11-25 2.6 Porcine pancreatic alpha-amylase in complex with helianthamide, a novel proteinaceous inhibitor

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