Book of Abstracts: Albany 2005
Domain Architecture in a Homodimeric Multi-substrate Enzyme
NAD+ synthetase catalyzes the last step in the biosynthesis of nicotinamide adenine dinucleotide. Magnesium divalent cations, potassium and ammonium monovalent cations are necessary for NAD+ synthetase activity. NAD+ synthetase is a symmetric homodimer with an extensive interface, 70% of which are stabilized by hydrophobic interactions, corresponding to about 20% of each monomer surface (1). Although the Bacillus subtilis NAD+ synthetase monomer consists of a single α/β domain, deamino-NAD+ does not bind in proximity to the βαβ motif at the C-terminal edge of the parallel β sheet. Instead, the deamino-NAD+-binding site is located at the subunit interface, where an extended cleft is defined by the quaternary structure of NAD synthetase (2). In the presence of salts, increasing the intra-molecular hydrophobic interactions, the dimer behaves hydrodynamically as a single ideal species and thermally unfolds according to a two-state dissociative mechanism (3). The presence of a small concentration of dimethyl sulfoxide destabilized the dimer and changed the unfolding mechanism of the enzyme. In order to determine whether or not surrounding mono- and divalent cations influence the homodimer conformation and to investigate the potential conformational changes in the NAD+ synthetase dimer interface, we have studied the thermally induced unfolding of the free NAD+ synthetase molecule in the absence of salts and in the presence of dimethyl sulfoxide by differential scanning calorimetry.
In this work we show that the thermal unfolding of the free NAD+ synthetase homodimer in the absence of cations is highly reversible and well approximated by two two-state transitions at (53.7 ± 0.1) °C and (56.5 ± 0.1) °C. We suggest that these transitions can be assigned to the unfolding of two ?energetic? domains of the homodimer that are stabilized by two major regions of the dimer interface which differ by the nature of the intermolecular contacts (4). The addition of dimethyl sulfoxide or polypropylene glycol results in the dissociation of the one domain of the homodimer, whereas the other domain unfolds as a dimer. Analytical size exclusion chromatography confirms the fact that the native NAD+ synthetase stays a dimer under the conditions studied. The NAD+ synthetase conformation of the homodimer with partially dissociated dimer interface can be important from the point of view of an inhibition mechanism study of this dimeric enzyme.
This research was supported by the NIH Grant U01056477.
References and Footnotes
Irina I. Protasevich*
Center for Biophysical Sciences and Engineering