Computational Studies of Glucoamylase Structure

Paper I

Coutinho, P.M. and Reilly, P.J. (1994), Structure-function relationships in the catalytic and starch-binding domains of glucoamylase, Protein Eng., 7, 393-400.

Sixteen primary sequences from five sub-families of fungal, yeast, and bacterial glucoamylases were related to structural information from the model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase obtained by protein crystallography. This domain is composed of thirteen alapha-helices, with five conserved regions defining the active site. Interactions between alpha-methyl maltoside and active-site residues were modeled, and the importance of these residues on the catalytic action of different glucoamylases was shown by their presence in each primary sequence. The overall structure of the starch-binding domain of some fungal glucoamylases was determined based on homology to the C-terminal domains of Bacillus cyclodextrin glucosyltransferases. Crystallography indicated that this domain contains six to eight b-strands, and homology allowed the attribution of a disulfide bridge in the glucoamylase starch-binding domain. Glucoamylase residues Thr525, Asn530, and Trp560, homologous to B. stearothermophilus cyclodextrin glucosyltransferase residues binding to maltose in the C-terminal domain, could be involved in raw-starch binding. The structure and length of the linker region between the catalytic and starch-binding domains in fungal glucoamylases can vary substantially, a further indication of the functional independence of the two domains.

Paper II

Coutinho, P.M. and Reilly, P.J. (1994), Structural similarities in glucoamylases by hydrophobic cluster analysis, Protein Eng., (in press).

The model of the catalytic domain of Aspergillus awamori var. X100 glucoamylase was related to fourteen other glucoamylase protein sequences belonging to five sub-families. Structural features of the different sequences were revealed by multisequence alignment following hydrophobic cluster analysis. The alignment agreed with the hydrophobic microdomains, normally conserved throughout evolution, evaluated from the three-dimensional model. Saccharomyces and Clostridium glucoamylases lack the alpha-helix exterior to the catalytic domain. A different catalytic base was found in the Saccharomyces glucoamylase sub-family. The starch-binding domain of fungal glucoamylases has identical structural features and substrate interacting residues as the C-terminal domain of models of Bacillus circulans cyclodextrin glucosyltransferases. Three putative N-glycosylation sites were found in the same turns in glucoamylases of different sub-families. O-glycosylation is present at different levels in the catalytic domain and in the linker between the catalytic and starch-binding domains.