Circular permutation of 5-aminolevulinate synthase: effect on folding, conformational stability, and structure.

The first and regulatory step of heme biosynthesis in mammals begins with the pyridoxal 5'-phosphate-dependent condensation reaction catalyzed by 5-aminolevulinate synthase. The enzyme functions as a homodimer with the two active sites at the dimer interface. Previous studies demonstrated that circular permutation of 5-aminolevulinate synthase does not prevent folding of the polypeptide chain into a structure amenable to binding of the pyridoxal 5'-phosphate cofactor and assembly of the two subunits into a functional enzyme. However, while maintaining a wild type-like three-dimensional structure, active, circularly permuted 5-aminolevulinate synthase variants possess different topologies. To assess whether the aminolevulinate synthase overall structure can be reached through alternative or multiple folding pathways, we investigated the guanidine hydrochloride-induced unfolding, conformational stability, and structure of active, circularly permuted variants in relation to those of the wild type enzyme using fluorescence, circular dichroism, activity, and size exclusion chromatography. Aminolevulinate synthase and circularly permuted variants folded reversibly; the equilibrium unfolding/refolding profiles were biphasic and, in all but one case, protein concentration-independent, indicating a unimolecular process with the presence of at least one stable intermediate. The formation of this intermediate was preceded by the disruption of the dimeric interface or dissociation of the dimer without significant change in the secondary structural content of the subunits. In contrast to the similar stabilities associated with the dimeric interface, the energy for the unfolding of the intermediate as well as the overall conformational stabilities varied among aminolevulinate synthase and variants. The unfolding of one functional permuted variant was protein concentration-dependent and had a potentially different folding mechanism. We propose that the order of the ALAS secondary structure elements does not determine the ability of the polypeptide chain to fold but does affect its folding mechanism.