Abstract
In Sulfolobus solfataricus, Sso, the ADP-ribosylating thermozyme is known to carry both auto- and heteromodification of target proteins via short chains of ADP-ribose. Here, we provide evidence that this thermoprotein is a multifunctional enzyme, also showing ATPase activity. Electrophoretic and kinetic analyses were performed using NAD+ and ATP as substrates. The results showed that ATP is acting as a negative effector on the NAD+-dependent reaction, and is also responsible for inducing the dimerization of the thermozyme. These findings enabled us to further investigate the kinetic of ADP-ribosylation activity in the presence of ATP, and to also assay its ability to work as a substrate. Moreover, since the heteroacceptor of ADP-ribose is the sulfolobal Sso7 protein, known as an ATPase, some reconstitution experiments were set up to study the reciprocal influence of the ADP-ribosylating thermozyme and the Sso7 protein on their activities, considering also the possibility of direct enzyme/Sso7 protein interactions. This study provides new insights into the ATP-ase activity of the ADP-ribosylating thermozyme, which is able to establish stable complexes with Sso7 protein.
Highlights
The eukatyotic-like poly(ADP-ribosyl)ation system in the thermophilic S. solfataricus was unique [1,2], until the discovery of poly(ADP-ribose) polymerase (PARP) gene orthologues in several prokaryotes [3]
It is well known that the group of 7 kDa proteins is highly conserved in all Sulfolobus species, identified with different acronima (Sso, Sac, Sul) [13,14], and they belong to a non-histone group, as compared with histone-like proteins found in other Archaea [13]
In order to assess the effect of ATP on PARPSso, the same kinetic was carried out by measuring PARPSso activity, either in the absence or in the presence of ATP (5 μM, 10 μM and 100 μM) at increasing NAD+ concentrations (Figure 1)
Summary
The eukatyotic-like poly(ADP-ribosyl)ation system in the thermophilic S. solfataricus was unique [1,2], until the discovery of poly(ADP-ribose) polymerase (PARP) gene orthologues in several prokaryotes [3]. It is well known that the group of 7 kDa proteins is highly conserved in all Sulfolobus species, identified with different acronima (Sso, Sac, Sul) [13,14], and they belong to a non-histone group, as compared with histone-like proteins found in other Archaea [13] The latter form a core which DNA wraps on, whereas Sso proteins interact with the minor grooves of DNA, leading to the condensation of nucleic acid [15]. The reconstitution of purified PARPSso with homogenous Sso in the presence of 32P-NAD+in the absence of DNA led to demonstrate that the 7 kDa protein increases the ADP-ribosylating activity of the thermozyme, functioning as an in vitro ADP-ribose acceptor [8]. We studied whether PARPSso and Sso ATPase activities could have a reciprocal influence, and the two proteins would exhibit direct protein-protein interactions
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