Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.


Abstract

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type. Study holds ProTherm entries: 11752 Extra Details: catalytic domain; N-glycosylation site; active site

Submission Details

ID: FN7bthe73

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:43 p.m.

Version: 1

Publication Details
Sauer J;Christensen T;Frandsen TP;Mirgorodskaya E;McGuire KA;Driguez H;Roepstorff P;Sigurskjold BW;Svensson B,Biochemistry (2001) Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger. PMID:11478902
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
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
1AC0 1997-07-07 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN COMPLEX WITH CYCLODEXTRIN, NMR, MINIMIZED AVERAGE STRUCTURE
1KUM 1996-07-11 GLUCOAMYLASE, GRANULAR STARCH-BINDING DOMAIN, NMR, MINIMIZED AVERAGE STRUCTURE
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
1GAH 1996-08-17 2.0 GLUCOAMYLASE-471 COMPLEXED WITH ACARBOSE
5GHL 2017-10-18 2.0 Crystal structure Analysis of the starch-binding domain of glucoamylase from Aspergillus niger
3GLY 1994-11-01 2.2 REFINED CRYSTAL STRUCTURES OF GLUCOAMYLASE FROM ASPERGILLUS AWAMORI VAR. X100
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
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
6FRV 2018-05-09 2.3 Structure of the catalytic domain of Aspergillus niger Glucoamylase
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
96.9 Glucoamylase P23176 AMYG_ASPKA
100.0 Glucoamylase P69328 AMYG_ASPNG
100.0 Glucoamylase P69327 AMYG_ASPAW
95.9 Glucoamylase P22832 AMYG_ASPSH