Structure of Aspergillus aculeatus ß-1,4-galactanase in complex with galactobiose.

ß-1,4-Galactanases are glycoside hydrolases that are involved in the degradation of pectin and belong to family 53 in the classification of glycoside hydrolases. Previous studies have elucidated the structures of several fungal and two bacterial galactanases, while biochemical studies have indicated differences in the product profiles of different members of the family. Structural studies of ligand complexes have to date been limited to the bacterial members of the family. Here, the first structure of a fungal galactanase in complex with a disaccharide is presented. Galactobiose binds to subsites -1 and -2, thus improving our understanding of ligand binding to galactanases.

Effect of mutations on the thermostability of Aspergillus aculeatus ß-1,4-galactanase.

New variants of ß-1,4-galactanase from the mesophilic organism Aspergillus aculeatus were designed using the structure of ß-1,4-galactanase from the thermophile organism Myceliophthora thermophila as a template. Some of the variants were generated using PROPKA 3.0, a validated pKa prediction tool, to test its usefulness as an enzyme design tool. The PROPKA designed variants were D182N and S185D/Q188T, G104D/A156R. Variants Y295F and G306A were designed by a consensus approach, as a complementary and validated design method. D58N was a stabilizing mutation predicted by both methods. The predictions were experimentally validated by measurements of the melting temperature (Tm ) by differential scanning calorimetry. We found that the Tm is elevated by 1.1 °C for G306A, slightly increased (in the range of 0.34 to 0.65 °C) for D182N, D58N, Y295F and unchanged or decreased for S185D/Q188T and G104D/A156R. The Tm changes were in the range predicted by PROPKA. Given the experimental errors, only the D58N and G306A show significant increase in thermodynamic stability. Given the practical importance of kinetic stability, the kinetics of the irreversible enzyme inactivation process were also investigated for the wild-type and three variants and found to be biphasic. The half-lives of thermal inactivation were approximately doubled in G306A, unchanged for D182N and, disappointingly, a lot lower for D58N. In conclusion, this study tests a new method for estimating Tm changes for mutants, adds to the available data on the effect of substitutions on protein thermostability and identifies an interesting thermostabilizing mutation, which may be beneficial also in other galactanases.

Activity of three ß-1,4-galactanases on small chromogenic substrates.

ß-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal ß-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl ß-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus ß-1,4-galactanase at pH 4.4 and for M. giganteus ß-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis ß-1,4-galactanase did not hydrolyze 4-nitrophenyl ß-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial ß-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis ß-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis ß-1,4-galactanase to bind 4-nitrophenyl -1,4-ß-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-ß-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-ß-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.