According to their fold, pyridoxal 5'-phosphate-dependent enzymes are grouped into five superfamilies. Fold Type I easily comprises the largest and most investigated group. The enzymes of this group have very similar 3D structures. Remarkably, the location of the cofactor in the active site, between the two domains that form a single subunit, is almost identical in all members of the group. Nonetheless, Fold Type I enzymes show very little sequence identity, raising the question as to which structural features determine the common fold. An important fold determinant appears to be the presence of three evolutionarily conserved clusters of hydrophobic contacts. A previous investigation, which used Escherichia coli serine hydroxymethyltransferase, a well characterized Fold Type I member, demonstrated the involvement of one of these clusters in the stability of the quaternary structure. The present study focuses on the role of the same cluster in the stability of the cofactor binding site. The investigation was carried out by equilibrium denaturation experiments on serine hydroxymethyltransferase forms in which the hydrophobic contact area of the cluster under study was reduced by site-directed mutagenesis. The results obtained show that the mutations clearly affected the process of pyridoxal 5'-phosphate dissociation induced by urea, reducing the stability of the cofactor binding site. We suggest that the third cluster promotes the formation of a bridging structural region that stabilizes the overall protein structure by connecting the two domains, shaping the cofactor binding site and participating in the formation of the quaternary structure.