Thermal Unfolding of Nucleoside Hydrolases from the Hyperthermophilic Archaeon Sulfolobus solfataricus: Role of Disulfide Bonds

Abstract

Nucleoside hydrolases are metalloproteins that hydrolyze the N-glycosidic bond of β-ribonucleosides, forming the free purine/pyrimidine base and ribose. We report the stability of the two hyperthermophilic enzymes Sulfolobus solfataricus pyrimidine-specific nucleoside hydrolase (SsCU-NH) and Sulfolobus solfataricus purine-specific inosineadenosine- guanosine nucleoside hydrolase (SsIAG-NH) against the denaturing action of temperature and guanidine hydrochloride by means of circular dichroism and fluorescence spectroscopy. The guanidine hydrochloride-induced unfolding is reversible for both enzymes as demonstrated by the analysis of the refolding process by activity assays and fluorescence measurements. The evidence that the denaturation of SsIAG-NH carried out in the presence of reducing agents proved to be reversible indicates that the presence of disulfide bonds interferes with the refolding process of this enzyme. Both enzymes are highly thermostable and no thermal unfolding transition can be obtained up to 108°C. SsIAG-NH is thermally denatured under reducing conditions (T(m)=93°C) demonstrating the contribution of disulfide bridges to enzyme thermostability.

Ribonucleoside Hydrolases–Structure, Functions, Physiological Role and Practical Uses

“…The enzymes are also stable towards reducing agents such as dithiothreitol (DTT). When exposed to 0.6 M DTT for an hour, the enzymatic activity drops by 40%, which confirms the presence of disulfide bonds which stabilize the structure in the enzyme [ 51 , 69 ].…”

“…An example of a reaction for this type of hydrolase is shown in Figure 2 . Hydrolases of this type are found in archaea [ 50 , 51 , 52 , 53 ], protozoa [ 3 , 5 , 12 , 13 , 14 ], some bacteria [ 20 ], fungi [ 30 , 33 ], and insects [ 36 ]. Hydrolases of the third type are nonspecific purine–pyrimidine hydrolases.…”

“…For example, the pyrimidine-specific and purine-specific hydrolases from Saccharolobus solfataricus (previously named Sulfolobus solfataricus ) have an optimum enzyme activity at 100 °C, with enzyme incubation at this temperature for 10 min causing a loss of 10% of activity. Incubation for 10 min at 110 °C results in a loss of 80% of enzymatic activity [ 50 , 51 , 69 ]. From a structural point of view, this high stability is due to the large number of Leu and Ile residues compared to other hydrolases, as well as the presence of disulfide bonds.…”

Every OGT Is Illuminated … by Fluorescent and Synchrotron Lights

“…An interesting feature, not present in other AGTs, is the C29-C31 S-S bridge of the N-terminal domain, which is an important structural element, contributing to the impressive thermal stability of Ss OGT ( Table 2 ; [ 30 ]). Although disulfide bonds are rare in intracellular mesophilic proteins, recent genomic and biochemical data show that they are present in intracellular proteins of hyperthermophilic archaea, thus suggesting a role for disulfide bonding in stabilizing at least some thermostable proteins [ 67 , 68 ].…”