Refined structure for the complex of acarbose with glucoamylase from Aspergillus awamori var. X100 to 2.4-A resolution.

To correlate structural features with glucoamylase properties, a structure-based multisequence alignment was constructed using information from catalytic and starch-binding domain models. The catalytic domain is composed of three hydrophobic folding units, the most labile and least hydrophobic of them being missing in the most stable glucoamylase. The role of O-glycosylation in stabilizing the most hydrophobic folding unit, the only one where thermostabilizing mutations with unchanged activity have been made, is described. Differences in both length and composition of interhelical loops are correlated with stability and selectivity characteristics. Two new glucoamylase subfamilies are defined by using homology criteria. Protein parsimony analysis suggests an ancient bacterial origin for the glucoamylase gene. Increases in length of the belt surrounding the active site, degree of O-glycosylation, and length of the linker probably correspond to evolutionary steps that increase stability and secretion levels of Aspergillus-related glucoamylases.

Glucoamylase structural, functional, and evolutionary relationships

The three-dimensional structure of the complex of 1-deoxynojirimycin with glucoamylase II-(471) from Aspergillus awamori var. X100 has been determined to 2.4-A resolution. The model includes residues corresponding to residues 1-471 of glucoamylase I from Aspergillus niger, two molecules of bound 1-deoxynojirimycin and 605 sites for water molecules. The crystallographic R factor from refinement is 0.119, and the root-mean-squared deviation in bond distances is 0.012 A. The inhibitor complex confirms the location of the active site in the packing void of the alpha/alpha-barrel as proposed by Aleshin et al. [Aleshin, A., Golubev, A., Firsov, L., & Honzatko, R. B. (1992) J. Biol. Chem. 267, 19291-19298]. One inhibitor molecule is associated with strong electron density and represents the principal site of interaction of 1-deoxynojirimycin with the enzyme. The other 1-deoxynojirimycin molecule is associated with weak electron density and therefore, probably represents a binding site of low affinity. Interactions of 1-deoxynojirimycin with the enzyme at its principal site involve Arg 45, Asp 55, Arg 305, and carbonyl 177. In addition, a water molecule (water 500) hydrogen bonds to Glu 400 and the 6-hydroxyl of 1-deoxynojirimycin and is at an approximate distance of 3.3 A from the "anomeric" carbon of the inhibitor. The structural arrangement of functional groups near the inhibitor molecule suggests that Glu 179 is a catalytic acid, Glu 400 a catalytic base, and water 500 the attacking nucleophile in the hydrolysis of maltooligosaccharides. The relevance of the X-ray work to proposed mechanisms of enzymatic hydrolysis of oligosaccharides is discussed.

Refined structure for the complex of 1-deoxynojirimycin with glucoamylase from Aspergillus awamori var. X100 to 2.4-A resolution.

Publisher Summary One of the major advances in the last part of the twentieth century in the understanding of starch-degrading enzymes resulted from the application of modern biochemical and biophysical techniques of protein sequence analysis, chemical modification of specific amino acid groups, x-ray crystallography, recombinant DNA cloning and sequencing, and site-directed mutagenesis. These techniques, combined with the enzymological studies, have given a much more complete understanding of how the enzyme protein structure provides specificity and performs catalysis. Enzymes involved in the breakdown of starch chains are primarily of four types: those that hydrolyze (1→4) α-D-glucosidic bonds (amylases); those that hydrolyze (1→6) α-D-glucosidic bonds (isoamylases); those that transfer (1→4) α-D-glucosidic bonds (glucanosyltransferases); and branching enzymes [α-(1→4)α-(1→6) transferases]. The amylases can be divided into three classes: the endoacting α-amylases; the exoacting α-amylases; and isoamylases. The α-amylases are distinguished by two features, the formation of products that have the α-configuration at the reducing end anomeric carbon of the newly formed products and an endomechanism of attacking the starch chain at glucose residues in the interior parts of the starch chain. In the exomechanism, the enzyme binds glucose residues at the non-reducing ends of the starch chains, and frequently produces products that have the α-configuration at the anomeric carbon of the newly formed reducing end of the products. The endomechanism of α-amylases rapidly fragments starch chains into smaller chains and thereby rapidly decreases the starch–iodine–iodide blue color and the viscosity, and endoacting amylases are, therefore, sometimes called liquefying amylases.

Enzymes and Their Action on Starch

Refined structure for the complex of d-gluco-dihydroacarbose with glucoamylase from Aspergillus awamori var. X100 to 2.2 Å resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase

K. N. Neustroev

Papers

Acid proteinase and the multiplicity of aspergillus awamori glucoamylase forms