Restoration of Catalytic Activity beyond Wild-Type Level in Glucoamylase from Aspergillus awamori by Oxidation of the Glu400→Cys Catalytic-Base Mutant to Cysteinesulfinic Acid

Abstract

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Novel Dextranase Catalyzing Cycloisomaltooligosaccharide Formation and Identification of Catalytic Amino Acids and Their Functions Using Chemical Rescue Approach

“…At the 3Ј-flanking region of 229 bp, no termination loop was found downstream of the stop codon (5Ј-5089 TAA 5091 -3Ј). The deduced amino acid sequence contained an N-terminal sequence of ANQEEKQSSQAGL-RALTVS and many internal sequences (data not shown) of Lys-C peptidase-digested peptides, indicating that a signal peptide was cleaved at the typical processing site between Ala 38 and Ala 39 (26) and that the isolated gene encoded PsDex protein. A BLAST homology search revealed PsDex to be a GH-66 enzyme family member without obvious similarity to GH-49 enzymes (27)(28)(29) (30 -32).…”

Replacement of the Catalytic Nucleophile Aspartyl Residue of Dextran Glucosidase by Cysteine Sulfinate Enhances Transglycosylation Activity

“…One peptide of 1629.6 Da was revealed to be Gly-187-Lys-201 by complete Edman degradation sequencing. The mass of this peptide was higher than its theoretical mass of 1596.8 Da by ϳ32 Da, indicating that Cys-194 was converted to cysteine sulfinate as reported previously for two inverting glycosidases (13,14). Masses of 17 peptides were assigned, and 35% of the entire sequence was covered.…”

“…The thiol group of Cys-194 of D194C-2CS was oxidized by KI (13,14). 2CS, as a control, was also treated under the same conditions.…”

“…Cysteine sulfinate substitutions of the catalytic carboxylates (general base catalyst) in inverting glycosidases have been reported (13,14). In the case of GH 15 glucoamylase (EC 3.2.1.3), replacement of the general base catalyst by cysteine sulfinate increased the catalytic activity (the mutant enzymes showed 160% of the activity of the wild type) (13).…”

“…Cysteine sulfinate substitutions of the catalytic carboxylates (general base catalyst) in inverting glycosidases have been reported (13,14). In the case of GH 15 glucoamylase (EC 3.2.1.3), replacement of the general base catalyst by cysteine sulfinate increased the catalytic activity (the mutant enzymes showed 160% of the activity of the wild type) (13). On the other hand, the mutant GH 6 cellulase harboring cysteine sulfinate in the position of the catalytic base showed only 52% of the wild type activity, but it had higher activity at low pH than the wild type cellulase (14).…”