Richard Griessler

Structure–function relationships for Schizophyllum commune trehalose phosphorylase and their implications for the catalytic mechanism of family GT-4 glycosyltransferases

The cDNA encoding trehalose phosphorylase, a family GT-4 glycosyltransferase from the fungus Schizophyllum commune, was isolated and expressed in Escherichia coli to yield functional recombinant protein in its full length of 737 amino acids. Unlike the natural phosphorylase that was previously obtained as a truncated 61 kDa monomer containing one tightly bound Mg2+, the intact enzyme produced in E. coli is a dimer and not associated with metal ions [Eis, Watkins, Prohaska and Nidetzky (2001) Biochem. J. 356, 757-767]. MS analysis of the slow spontaneous conversion of the full-length enzyme into a 61 kDa fragment that is fully active revealed that critical elements of catalysis and specificity of trehalose phosphorylase reside entirely in the C-terminal protein part. Intact and truncated phosphorylases thus show identical inhibition constants for the transition state analogue orthovanadate and alpha,alpha-trehalose (K(i) approximately 1 microM). Structure-based sequence comparison with retaining glycosyltransferases of fold family GT-B reveals a putative active centre of trehalose phosphorylase, and results of site-directed mutagenesis confirm the predicted crucial role of Asp379, His403, Arg507 and Lys512 in catalysis and also delineate a function of these residues in determining the large preference of the wild-type enzyme for the phosphorolysis compared with hydrolysis of alpha,alpha-trehalose. The pseudo-disaccharide validoxylamine A was identified as a strong inhibitor of trehalose phosphorylase (K(i)=1.7+/-0.2 microM) that displays 350-fold tighter binding to the enzyme-phosphate complex than the non-phosphorolysable substrate analogue alpha,alpha-thio-trehalose. Structural and electronic features of the inhibitor that may be responsible for high-affinity binding and their complementarity to an anticipated glucosyl oxocarbenium ion-like transition state are discussed.

Efficient downstream processing of maltodextrin phosphorylase from Escherichia coli and stabilization of the enzyme by immobilization onto hydroxyapatite

Downstream processing by biospecific chromatography of maltodextrin phosphorylase from Escherichia coli, overexpressed in E. coli, was substantially improved by a novel approach using ceramic hydroxyapatite. Wild-type and a less active mutant enzyme were purified from crude bacterial cell extracts in one efficient separation step that yielded phosphorylase in purity > 95% in at least 90% recoveries. At pH 6.9 and 25 degrees C, wild-type and mutant phosphorylases eluted from the hydroxyapatite column at a phosphate concentration of 0.4 M whereas calcium ions failed to displace the enzymes. The dynamic capacity for phosphorylase binding in the presence of bulk proteins was approximately 3 mg enzyme ml-1 matrix. The interaction of E. coli phosphorylase with hydroxyapatite seems to be mediated by surface amino groups, so that the bound enzyme retained almost full catalytic activity. Compared to the soluble enzyme, immobilization onto hydroxyapatite resulted in a more than 30-fold stabilization of wild-type phosphorylase against thermal and proteolytic inactivation and thus could improve the operational stability of phosphorylase during conversion of polysaccharide to glucose 1-phosphate.

Reaction engineering aspects of alpha-l,4-D-glucan phosphorylase catalysis : comparison of plant and bacterial enzymes for the continuous synthesis of D-glucose-1-phosphate.

Some important process properties of alpha-l,4-D-glucan phosphorylases isolated from the bacterium Corynebacterium callunae and potato tubers (Solatium tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (K (M) of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300-400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15-20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40 degrees C was identified.Some important process properties of α-l,4-D-ghican phosphorylases isolated from the bacteriumCorynebacterium callunae and potato tubers (Solatium tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (KM of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300–400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15–20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40°C was identified.

Reaction Engineering Aspects of α-1,4- d -Glucan Phosphorylase Catalysis

Some important process properties of α-1,4-d-glucan phosphorylases isolated from the bacterium Corynebacterium callunae and potato tubers (Solanum tuberosum) were compared. Apart from minor differences in their stability and specificity (represented by the maximum degree of maltodextrin conversion) and a 10-fold higher affinity of the plant phosphorylase for maltodextrin (K M of 1.3 g/L at 300 mM of orthophosphate), the performances of both enzymes in a continuous ultrafiltration membrane reactor were almost identical. Product synthesis was carried out over a time course of 300–400 h in the presence or absence of auxiliary pullulanase (increasing the accessibility of the glucan substrate for phosphorolytic attack up to 15–20%). The effect of varied dilution rate and reaction temperature on the resulting productivities was quantitated, and a maximum operational temperature of 40°C was identified.