Aspartate aminotransferase (AspAT) from alkalophilic Bacillus circulans contains an additional N-terminal sequence of 32 amino acid residues that are absent in all other AspATs from different sources. Modeling suggested that this sequence forms two alpha-helical segments which establish a continuous network of interactions on the surface of the molecule. In the present study, we studied the role of the N-terminal sequence in folding and stability of AspAT by applying the scanning calorimetry, and CD and fluorescence spectroscopies to the native and truncated enzymes. Truncated AspAT (Delta2alpha mutant) devoid of N-terminal residues cannot provide sufficient potential of quaternary intersubunit and subunit-cofactor interactions, which results in a monomeric non-functional conformation. However, the residual tertiary interactions in the Delta2alpha mutant are sufficient to: i) provide stability of a residual structure over a wide pH range; ii) confer moderate cooperativity of the denaturant-induced transition while only low cooperativity of the thermal transition, and iii) maintain the hydrophobic core of a part of the structure which prevents aromatic fluorophores from quenching by water. Furthermore, the present study provides evidence that AspAT from the alkalophilic bacterium follows unfolding pathway comprising a stable non-functional intermediate, in contrast to a two-state mechanism of the thermophilic AspAT from Sulfolobus solfataricus.