Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase.


Abstract

To decrease irreversible thermoinactivation of Aspergillus awamori glucoamylase, five Gly residues causing helix flexibility were replaced with Ala residues. Mutation of Gly57 did not affect thermostability. Mutation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it at pH 5.5. The Gly139-->Ala mutation did not change thermostability at pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5. The Gly 137/Gly139-->Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at pHs 3.5-5.5. Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases are the most stable yet produced by mutation. Guanidine treatment at pH 4.5 decreased the reversible stabilities of Gly137-->Ala, Gly139-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases at infinite dilution while not changing those of Gly251-->Ala and Gly383-->Ala glucoamylases, which is, in general, opposite to what occurred with thermoinactivation. Mutation of Gly57 greatly improved the extracellular glucoamylase production by yeast, that of Gly137 barely affected it and those of Gly139 and of both Gly137 and Gly139 strongly impeded it. These observations suggest that alpha-helix rigidity can affect reversible and irreversible glucoamylase stability differently, that the effects of multiple mutations within one alpha-helix to improve stability are not always additive and that even single mutations can strongly affect extracellular enzyme production. Study holds ProTherm entries: 11250, 11251, 11252, 11253, 11254, 11255 Extra Details: alpha-helix; alanine; glucoamylase; glycine;,guanidine; production; thermostability

Submission Details

ID: gS6owQfy

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
Chen HM;Li Y;Panda T;Buehler FU;Ford C;Reilly PJ,Protein Eng. (1996) Effect of replacing helical glycine residues with alanines on reversible and irreversible stability and production of Aspergillus awamori glucoamylase. PMID:8862550
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
1KUM 1996-07-11 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN, NMR, MINIMIZED AVERAGE STRUCTURE
1AC0 1997-07-07 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN COMPLEX WITH CYCLODEXTRIN, NMR, MINIMIZED AVERAGE STRUCTURE
1ACZ 1997-07-07 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN COMPLEX WITH CYCLODEXTRIN, NMR, 5 STRUCTURES
1KUL 1996-07-11 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN, NMR, 5 STRUCTURES
1GAI 1996-08-17 1.7 GLUCOAMYLASE-471 COMPLEXED WITH D-GLUCO-DIHYDROACARBOSE
3EQA 2009-10-13 1.9 Catalytic domain of glucoamylase from aspergillus niger complexed with tris and glycerol
5GHL 2017-10-18 2.0 Crystal structure Analysis of the starch-binding domain of glucoamylase from Aspergillus niger
1GAH 1996-08-17 2.0 GLUCOAMYLASE-471 COMPLEXED WITH ACARBOSE
3GLY 1994-11-01 2.2 REFINED CRYSTAL STRUCTURES OF GLUCOAMYLASE FROM ASPERGILLUS AWAMORI VAR. X100
6FRV 2018-05-09 2.3 Structure of the catalytic domain of Aspergillus niger Glucoamylase
1DOG 1994-01-31 2.3 REFINED STRUCTURE FOR THE COMPLEX OF 1-DEOXYNOJIRIMYCIN WITH GLUCOAMYLASE FROM (ASPERGILLUS AWAMORI) VAR. X100 TO 2.4 ANGSTROMS RESOLUTION
1AGM 1994-09-30 2.3 Refined structure for the complex of acarbose with glucoamylase from Aspergillus awamori var. x100 to 2.4 angstroms resolution
1GLM 1994-06-22 2.4 REFINED CRYSTAL STRUCTURES OF GLUCOAMYLASE FROM ASPERGILLUS AWAMORI VAR. X100

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
99.3 Glucoamylase P22832 AMYG_ASPSH
95.1 Glucoamylase P69328 AMYG_ASPNG
95.1 Glucoamylase P69327 AMYG_ASPAW
100.0 Glucoamylase I P23176 AMYG_ASPKA