Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima

Background: Oligomeric proteins may have been selected for in hyperthermoph iles because subunit association provides extra stabilization. Phosphoribos ylanthranilate isomerase (PRAI) is monomeric and labile in most mesophilic microorganisms, but dimeric and stable in the hyperthermophile Thermotoga m aritima (tPRAI). The two subunits of tPRAI are associated back-to-back and are locked together by a hydrophobic loop. The hypothesis that dimerization is important for thermostability has been tested by rationally designing m onomeric variants of tPRAI. Results: The comparison of tPRAI and PRAI from Escherichia coli (ePRAI) sug gested that levelling the nonplanar dimer interface would weaken the associ ation. The deletion of two residues in the loop loosened the dimer. Subsequ ent filling of the adjacent pocket and the exchange of polar for apolar res idues yielded a weakly associating and a nonassociating monomeric variant. Both variants are as active as the parental dimer but far more thermolabile . The thermostability of the weakly associating monomer increased significa ntly with increasing protein concentration. The X-ray structure of the nona ssociating monomer differed from that of the parental subunit only in the r estructured interface. The orientation of the original subunits was maintai ned in a crystal contact between two monomers. Conclusions: tPRAI is dimeric for reasons of stability. The clearly separat ed responsibilities of the beta alpha loops, which are involved in activity , and the ap loops, which are involved in protein stability, has permitted the evolution of dimers without compromising their activity. The preserved interaction in the crystal contacts suggests the most likely model for dime r evolution.