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The enzyme serine hydroxymethyltransferase (SHMT) plays a key role in folate metabolism and is conserved in all kingdoms of life.  SHMT is a pyridoxal 5’-phosphate (PLP) - dependent enzyme that catalyzes the conversion of L-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. Crystal structures of multiple members of the SHMT family have shown that the enzyme has a single conserved cis proline, which is located near the active site.  Here, we have characterized a Pro to Ser amino acid variant (P285S) that affects this conserved cis proline in soybean SHMT8.  P285S was identified as one of a set of mutations that affect the resistance of soybean to the agricultural pathogen soybean cyst nematode.  We find that replacement of Pro285 by serine eliminates PLP-mediated catalytic activity of SHMT8, reduces folate binding, decreases enzyme stability, and affects the dimer-tetramer ratio of the enzyme in solution.  Crystal structures at 1.9 – 2.2 Å resolution reveal a local reordering of the polypeptide chain that extends an a-helix and shifts a turn region into the active site.  This results in a dramatically perturbed PLP-binding pose, where the ring of the cofactor is flipped by ~180° with concomitant loss of conserved enzyme-PLP interactions.  A nearby region of the polypeptide becomes disordered, evidenced by missing electron density for ~10 residues.  These structural perturbations are consistent with the loss of enzyme activity and folate binding and underscore the important role of the Pro285 cis-peptide in SHMT structure and function.