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.

Relationships between structure, function and stability for pyridoxal 5'-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies.

Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5'-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 degrees C was, respectively, four- and threefold reduced in two mutants, Arg234-->Ala and Arg242-->Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem.270, 2126-2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5'-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234-->Ala and Arg242-->Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K(app)), compared to the wild-type (K(app) approximately 6 mm). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but approximately 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK(a) values for the cofactor 5'-phosphate and the exogenous oxyanion.

Mutagenesis of the dimer interface region of Corynebacterium callunae starch phosphorylase perturbs the phosphate-dependent conformational relay that enhances oligomeric stability of the enzyme.

We have used alanine-scanning site-directed mutagenesis of the dimer contact region of starch phosphorylase from Corynebacterium callunae to explore the relationship between a protein conformational change induced by phosphate binding and the up to 500-fold kinetic stabilization of the functional quarternary structure of this enzyme when phosphate is present. Purified mutants (at positions Ser-224, Arg-226, Arg-234, and Arg-242) were characterized by Fourier transform-infrared (FT-IR) spectroscopy and enzyme activity measurements at room temperature and under conditions of thermal denaturation. Difference FT-IR spectra of wild type and mutants in (2)H(2)O solvent revealed small changes in residual amide II band intensities at approximately 1,550 cm(-1), indicating that (1)H/(2)H exchange in the wild type is clearly perturbed by the mutations. Decreased (1)H/(2)H exchange in comparison to wild type suggests formation of a more compact protein structure in S224A, R234A, and R242A mutants and correlates with rates of irreversible thermal denaturation at 45 degrees C that are up to 10-fold smaller for the three mutants than the wild type. By contrast, the mutant R226A inactivates 2.5-fold faster at 45 degrees C and shows a higher (1)H/(2)H exchange than the wild type. Phosphate (20 mM) causes a greater change in FT-IR spectra of the wild type than in those of S224A and 234A mutants and leads to a 5-fold higher stabilization of the wild type than the two mutants. Therefore, structural effects of phosphate binding leading to kinetic stability of wild-type starch phosphorylase are partially complemented in the S224A and R234A mutants. Infrared spectroscopic measurements were used to compare thermal denaturations of the mutants and the wild type in the absence and presence of stabilizing oxyanion. The broad denaturation transition of unliganded wild type in the range 40-50 degrees C is reduced in the S224A and R234A mutants, and this reflects mainly a shift of the onset of denaturation to a 4-5 degrees C higher value.

Tracking interactions that stabilize the dimer structure of starch phosphorylase from Corynebacterium callunae. Roles of Arg234 and Arg242 revealed by sequence analysis and site-directed mutagenesis.

Glycogen phosphorylases (GPs) constitute a family of widely spread catabolic alpha1,4-glucosyltransferases that are active as dimers of two identical, pyridoxal 5'-phosphate-containing subunits. In GP from Corynebacterium callunae, physiological concentrations of phosphate are required to inhibit dissociation of protomers and cause a 100-fold increase in kinetic stability of the functional quarternary structure. To examine interactions involved in this large stabilization, we have cloned and sequenced the coding gene and have expressed fully active C. callunae GP in Escherichia coli. By comparing multiple sequence alignment to structure-function assignments for regulated and nonregulated GPs that are stable in the absence of phosphate, we have scrutinized the primary structure of C. callunae enzyme for sequence changes possibly related to phosphate-dependent dimer stability. Location of Arg234, Arg236, and Arg242 within the predicted subunit-to-subunit contact region made these residues primary candidates for site-directed mutagenesis. Individual Arg-->Ala mutants were purified and characterized using time-dependent denaturation assays in urea and at 45 degrees C. R234A and R242A are enzymatically active dimers and in the absence of added phosphate, they display a sixfold and fourfold greater kinetic stability of quarternary interactions than the wild-type, respectively. The stabilization by 10 mm of phosphate was, however, up to 20-fold greater in the wild-type than in the two mutants. The replacement of Arg236 by Ala was functionally silent under all conditions tested. Arg234 and Arg242 thus partially destabilize the C. callunae GP dimer structure, and phosphate binding causes a change of their tertiary or quartenary contacts, likely by an allosteric mechanism, which contributes to a reduced protomer dissociation rate.