Scanning tunnelling microscopy of Aspergillus niger glucoamylases

Abstract

The primary structure of glucoamylase 1 (glucan-1,4-α-glucosidase, EC 3.2.1.3) from Aspergillus niger consists of a catalytic and a binding domain separated by an O-glycosylated peptide 40 amino acid residues in length. Scanning tunnelling microscopic (STM) images of glucoamylase 2 (the catalytic domain with linker peptide) and a proteolytically cleaved product (the binding domain G1C, residues 471–616 of glucoamylase 1) confirmed that these proteins are globular and nearly spherical. Observed dimensions are: isolated catalytic domain (median half-height diameter, d_m= 5.8 nm, ‘in-plane’ axial ratio, γ= 1.15 : 1), isolated binding domain (d_m= 2.2 nm, γ= 1.18 : 1), catalytic domain within glucoamylase 1 (d_m= 5.9 nm, γ= 1.1 : 1) and binding domain within glucoamylase 1 (d_m= 3.4 nm, γ= 1.1 : 1). However, STM images of glucoamylase 1 suggest that the enzyme consists of two spatially separated globular domains. Measurements of the inter-domain spacing (median spacing between domain centres in glucoamylase 1, d_s= 9.5 nm) suggest that the linker glycopeptide is extended and semi-rigid. These data suggest that the O-glycosylated sequence may play an important role in determining the shape and functionality of the enzyme.