H.L. Bagger; C.C. Fuglsang; P. Westh. (2003)
"Preferential binding of two compatible solutes to the glycan moieties of Peniophora Lycii phytase."
Biochemistry, 42, 10295-10300 (2003)
Abstract
Regulation of hydration behavior, and the concomitant effects on solubility and other properties, has been suggested as a main function of protein glycosylation. In this work, we have studied the hydration of the heavily glycosylated Peniophora lycii phytase in solutions (0.15-1.1 m) of the two compatible solutes glycerol and sorbitol. Osmometric measurements showed that glycerol preferentially binds to phytase (i.e., glycerol-glycoprotein interactions are more favorable than water-glycoprotein interactions resulting in a preferential accumulation of glycerol near the protein interface), while sorbitol is preferentially excluded from the hydration sphere (water-glycoprotein interactions are the more favorable). To assess contributions from carbohydrate and peptide moieties, respectively, we compared phytase (Phy) and a modified, yet enzymatically active form (dgPhy) in which 90% of the glycans had been removed. This revealed that both polyols showed a pronounced and approximately equal degree of preferential binding to the carbohydrate moiety. This preferential binding of polyols to glycans is in contrast to the exclusion from peptide interfaces observed here (for dgPhy) and in numerous previous reports on nonglycosylated proteins. Despite the distinct differences between peptide and carbohydrate groups, glycosylation had no effect on the stabilizing action provided by glycerol and sorbitol. On the basis of this, it was concluded that the carbohydrate mantle of Phy is equally accessible in the native and thermally denatured states, respectively (most likely fully accessible in both), and thus that its interactions with compatible solutes have little or no effect on conformational equilibria of the glycoprotein. For solubility and aggregation equilibria, on the other hand, the results suggest a polyol-induced stabilization of monomeric forms.
A.D. Nielsen; C.C. Fuglsang; P. Westh.
"Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation."
Biochem. J., 373, 337-343 (2003)
Abstract
The effect of temperature and calcium ions on the denaturation of a recombinant á-amylase from Bacillus halmapalus á-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature (Td) of 83 °C in the presence of excess calcium ions was observed, whereas Td decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy EA of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calciu m-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent EA can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.
A.D. Nielsen; M.L. Pusey; C.C. Fuglsang; P. Westh.
"A proposed mechanism for the thermal denaturation of a recombinant Bacillus halmapalus alpha-amylase ? the effect of calcium ions."
Biochim. Biophys. Acta., 1652, 52-63 (2003)
Abstract
The thermal stability of a recombinant a-amylase from Bacillus halmapalus alpha-amylase (BHA) has been investigated using circular dichroism spectroscopy (CD) and differential scanning calorimetry (DSC). This alpha-amylase is homologous to other Bacillus alpha-amylases where crystallographic studies have identified the existence of three calcium binding sites in the structure. Denaturation of BHA is irreversible with a T-m of approximately 89 degreesC and DSC thermograms can be described using a one-step irreversible model. A 5 degreesC increase in T-m in the presence of 10- fold excess CaCl2 was observed. However, a concomitant increase in the tendency to aggregate was also observed. The presence of 30-40-fold excess calcium chelator (ethylenediaminetetraacetic acid (EDTA) or ethylene glycol- bis[beta-aminoethyl ether] N, N, N', N'-tetraacetic acid (EGTA)) results in a large destabilization of BHA, corresponding to about 40 degreesC lower T-m as deter mined by both CD and DSC. Ten-fold excess EGTA reveals complex DSC thermograms corresponding to both reversible and irreversible transitions, which probably originate from different populations of BHA/calcium complexes. Combined interpretation of these observations and structural information on homologous a-amylases forms the basis for a suggested mechanism underlying the inactivation mechanism of BHA. The mechanism includes irreversible thermal denaturation of different BHA/calcium complexes and the calcium binding equilibria. Furthermore, the model accounts for a temperature-induced reversible structural change associated with calcium binding.
F. Xu; E.J. Golightly; K.R. Duke; S.F. Lassen; B. Knusen; S. Christensen; K.M. Brown; S.H. Brown; M. Schulein.
"Humicola insolens cellobiose dehydrogenase: cloning, redox chemistry, and "logic gate"-like dual functionality."
Enzyme and Microbial Technology, 28(9-10), 744-753 (2001)
Abstract
Cellobiose dehydrogenase is a hemoflavoenzyme that catalyzes the sequential electron-transfer from an electron-donating substrate (e.g. cellobiose) to a flavin center, then to an electron-accepting substrate (e.g. quinone) either directly or via a heme center after an internal electron-transfer from the flavin to heme. We cloned the dehydrogenase from Humicola insolens, which encodes a protein of 761 amino acid residues containing an N-terminal heme domain and a C- terminal Ravin domain, and studied how the catalyzed electron transfers are regulated. Based on the correlation between the rate and redox potential, we demonstrated that with a reduced flavin center, the enzym e, as a reductase, could export electron from its heme center by a "outer-sphere" mechanism. With the "resting" flavin center, however, the enzyme could have a peroxidase-like function and import electron to its heme center after a peroxidative activation. The dual functionality of its heme center makes the enzyme a molecular "logic gate", in which the electron how through the heme center can be switched in direction by the redox state of the coupled flavin center.
C.C. Fuglsang; R.M. Berka; J.A. Wahleithner; S. Kauppinen; J.R. Shuster; G. Rasmussen; T. Halkier,; H. Dalbøge B. Henrissat.
"Biochemical analysis of recombinant fungal mutanases."
J. Biol. Chem. 275, 2009-2018 (2000)
Abstract
Nucleotide sequence analysis shows that Trichoderma harzianum and Penicillium purpurogenum 1,3-glucanases (mutanases) have homologous primary structures (53% amino acid sequence identity), and are composed of two distinct domains: a NH2-terminal catalytic domain and a putative COOH-terminal polysaccharide-binding domain separated by a O-glycosylated Pro-Ser-Thr-rich linker peptide. Each mutanase was expressed in Aspergillus oryzae host under the transcriptional control of a strong -amylase gene promoter. The purified recombinant mutanases show a pH optimum in the range from pH 3.5 to 4.5 and a temperature optimum around 50-55 °C at pH 5.5. Also, they exhibit strong binding to insoluble mutan with KD around 0.11 and 0.13 °M at pH 7 for the P. purpurogenum and T. harzianum mutanases, respectively. Partial hydrolysis showed that the COOH-terminal domain of the T. harzianum mut anase binds to mutan. The catalytic domains and the binding domains were assigned to a new family of glycoside hydrolases and to a new family of carbohydrate-binding domains, respectively.