Circular permutation of 5-aminolevulinate synthase: Mapping the polypeptide chain to its function

5-Aminolevulinate synthase (EC 2.3.1.37) is the first enzyme in the heme biosynthetic pathway. It catalyzes a condensation of glycine and succinyl-CoA to form aminolevulinic acid, CoA-SH and CO₂. This is the rate limiting and major regulatory step of heme biosynthesis in mammals and some bacteria. Recent site-directed mutagenesis studies of 5-aminolevulinate synthase identified amino acid residues involved in catalysis, substrate or cofactor binding. Although the roles of specific amino acid residues have been elucidated, much less is known about the involvement of 5-aminolevulinate synthase polypeptide chain arrangement in its folding, structure and function. To assess the importance of continuity of the 5-aminolevulinate synthase polypeptide chain, either random or engineered circularly permuted variants have been constructed. The circular permutation disrupts the continuity of a protein polypeptide chain by placing N- and C-termini into new locations without altering the amino acid composition of the protein. cDNA sequence analysis of the created active circularly permuted variants has shown that 5-aminolevulinate synthase is able to tolerate disruption of its polypeptide chain by circular permutation without loss of its activity. The central part of the protein, containing the amino acids previously shown to be crucial for cofactor binding and catalysis, was found to be intolerant to disruption. Four of the active circularly permuted variants were purified to homogeniosity and biochemically characterized. This allowed identification of two functional elements, with roles in catalysis and glycine binding. The functional element is defined as a continuous stretch of polypeptide chain for which integrity is required to maintain appropriate enzymatic function. The effects of circular permutation on the structure of 5-aminolevulinate synthase were further assessed using denaturant-induced equilibrium unfolding studies. The thermodynamic stabilities suggested that circular permutation primarily affected the folding of individual subunits while formation of the dimer interface remained similar among variants. The change in subunit folding was translated into non-wild type topologies of active sites as confirmed by cofactor fluorescence quenching and homology modeling studies. The homology modeling analysis also suggested that the two functional elements identified are defined structural domains of 5-aminolevulinate synthase, connected by 0an unstructured loop. However, these domains cannot be considered as autonomous folding units because swapping them modifies both the stability and folding kinetics of the protein.