Role of proline residues in the folding of serine hydroxymethyltransferase

Abstract

Previous studies on the folding mechanism of Escherichia coli serine hydroxymethyltransferase (SHMT) showed that the final rate determining folding step was from an intermediate that contained two fully folded domains with N-terminal segments of approximately 55 residues and interdomain segments of approximately 50 residues that were still solvent exposed and subject to proteolysis. The interdomain segment contains 3 Pro residues near its N terminus and 2 Pro residues near its C terminus. The 5 Pro residues were each mutated to both a Gly and Ala residue, and each mutant SHMT was purified and characterized with respect to kinetic properties, stability, secondary structure, and folding mechanism. The results showed that Pro214 and Pro218 near the N terminus of the interdomain segment are not critical for folding, stability, or activity. The P216A mutant also retained most of the characteristics of the native enzyme, but its folding rate was altered. However, the P216G mutant was severely compromised in folding into a catalytically competent enzyme. Mutation of both Pro258 and Pro 264 had altered folding kinetics and resulted in enzymes that expressed little catalytic activity. The Phe257-Pro258 bond is cis in its configuration, and the P258A mutant SHMT showed reduced thermal stability. Pro216, Pro258, and Pro264 are conserved in all 53 known sequences of this enzyme. The results are discussed in terms of the role of each Pro residue in maintaining the structure and function of SHMT and a possible role in pyridoxal 5′-phosphate addition to the apo-enzyme.