Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase.

Structure of glucoamylase from Saccharomycopsis fibuligera at 1.7 A resolution.

The yeast Saccharomycopsis fibuligera produces a glucoamylase which belongs to sequence family 15 of glycosyl hydrolases. The structure of the non-glycosyl-ated recombinant enzyme has been determined by molecular replacement and refined against 1.7 A resolution synchrotron data to an R factor of 14.6%. This is the first report of the three-dimensional structure of a yeast family 15 glucoamylase. The refinement from the initial molecular-replacement model was not straightforward. It involved the use of an unrestrained automated refinement procedure (uARP) in combination with the maximum-likelihood refinement program REFMAC. The enzyme consists of 492 amino-acid residues and has 14 alpha-helices, 12 of which form an (alpha/alpha)6 barrel. It contains a single catalytic domain but no starch-binding domain. The fold of the molecule and the active site are compared to the known structure of the catalytic domain of a fungal family 15 glucoamylase and are shown to be closely similar. The active- and specificity-site residues are especially highly conserved. The model of the acarbose inhibitor from the analysis of the fungal enzyme fits tightly into the present structure. The active-site topology is a pocket and hydrolysis proceeds with inversion of the configuration at the anomeric carbon. The enzyme acts as an exo-glycosyl hydrolase. There is a Tris [2-amino-2-(hydroxymethyl)-1,3-propanediol] molecule acting as an inhibitor in the active-site pocket.

Crystallization and preliminary X-ray analysis of the Saccharomycopsis fibuligera glucoamylase expressed from the GLU1 gene in Escherichia coli.

The active non-glycosylated glucoamylase, overexpressed from the Saccharomycopsis fibuligera GLU1 gene in Escherichia coli BL21(DE3), has been purified from the solubilized inclusion bodies and then renatured in vitro. Crystals of the recombinant glucoamylase were obtained by vapour diffusion using PEG as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions of a = 58.1, b = 87.8 and c = 99.9 A, and diffract to 1.7 A resolution. This is the first report of the crystallization of the full-length glucoamylase corresponding to the mature enzyme.

High-yield production of Saccharomycopsis fibuligera glucoamylase in Escherichia coli, refolding, and comparison of the nonglycosylated and glycosylated enzyme forms.

The truncated GLA1 gene encoding the mature form of glucoamylase from the yeast Saccharomycopsis fibuligera has been over-expressed in Escherichia coli using the IPTG inducible pET system. Over-expression has led to the accumulation of insoluble glucoamylase in inclusion bodies from which an electrophoretically homogeneous active enzyme has been prepared yielding 30 mg per litre medium. This protein represents an N-terminus Met-free, non-glycosylated product which displays the identical specific activity of 45 units/mg and reduced thermal stability when compared to glycosylated enzymes isolated from Saccharomyces cerevisiae carrying the GLA1 gene. These data suggest that S. cerevisiae glycosylation of S. fibuligera glucoamylase does not play a critical role in enzymatic activity but that it does contribute to its thermal stability.