Structure-function studies of processing alpha-glucosidase-i

Processing α-glucosidase-I (Glu-I) is an endoplasmic reticulum inner membrane-bound enzyme that plays a critical role in N-glycosylation and quality-control of protein-folding. Despite its role, details of the catalytic mechanism of Glu-I function are still unknown. The objective of this research was to address the main obstacles in studying this enzyme, namely the lack of sufficient quantities of relatively pure enzyme and a substrate to test its function. Initially, a soluble form of yeast Glu-I was expressed in Escherichia coli with a yield of 6-8 mg of Glu-I per litre of culture. After single-step purification using immobilised metal affinity chromatography, this recombinant 6xHis-tagged Glu-I showed a Km of 1.27 mM with the synthetic trisaccharide substrate α-D-Glc1,2α-D-Glc1,3α-D-Glc-O-CH3. Since the catalytic domain of Glu-I is located at the C-terminus, expression of the C-terminal domain (Cwh41Δ1-525p) was attempted, but yielded insoluble bodies. Expression of Cwh41Δ1-525p with solubility-enhancing fusion tags or the co-expression of molecular chaperones did not improve solubility. Subsequently, based on a published tertiary structure of Glu-I, I identified that Cwh41Δ1-525p lacks two α-helices of the catalytic (α/α)6 toroid domain. Therefore, the N-terminus of Cwh41Δ1-525p was extended to include the missing helices and expression of the two new constructs (Cwh41Δ1-349p and Cwh41Δ1-314p) was attempted. However, these proteins also expressed as insoluble bodies. Co-expression of the N-terminal domain (Cwhnp) improved the expression of soluble Cwh41Δ1-525p, but the expressed protein was not functional. Catalytic domain released by trypsin hydrolysis from Glu-I was 2.2 times more active than the intact Glu-I. This catalytic domain was purified using size-exclusion chromatography. Since the enzymatic hydrolysis of a glycosidic bond typically occurs with general acid and general base assistance from two amino acid side chains, generally carboxylic amino acids, site-directed mutagenesis of all six conserved carboxylic residues of the catalytic domain was carried out. Glutamic acid 804 was identified as the catalytic base with the aid of nucleophile rescue. Further studies on the structure-function of yeast Glu-I will be helpful in establishing a model to study the inborn errors of metabolism involving this enzyme (CDG-IIb) in humans.