Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode

Abstract

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5′-phosphate–dependent enzyme that converts l-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Here, we determined five crystal structures of the 1884-residue SHMT8 tetramers from the SCN-susceptible cultivar (cv.) Essex and the SCN-resistant cv. Forrest (whose resistance is derived from the SHMT8 polymorphisms in Peking); the crystal structures were determined in complex with various ligands at 1.4–2.35 Å resolutions. We find that the two Forrest-specific polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the (6S)-tetrahydrofolate–binding site. Ligand-binding and kinetic studies indicate severely reduced affinity for folate and dramatically impaired enzyme activity in Forrest SHMT8. These findings imply widespread effects on folate metabolism in soybean cv. Forrest that have implications for combating the widespread increase in virulent SCN.

Mutations at the Serine Hydroxymethyltransferase Impact Its Interaction with a Soluble NSF Attachment Protein and a Pathogenesis-Related Protein in Soybean

“…Any disruption of this area may lead to loss of tetramerization (Jagath et al, 1997a). Therefore, our data support the idea that, at least in soybean, the cytosolic GmSHMT08 is present as a tetramer, as has been suggested earlier [ 9 , 10 , 29 , 52 ]. Moreover, mutations affecting the GmSHMT08 subunits, including dimerization and tetramerization residues, negatively impacted the GmSNAP18/GmSHMT08/GmPR08-Bet VI multi-protein complex interaction, suggesting that the presence of the GmSHMT08 tetramer is essential for the multi-protein complex.…”

“…In the current study, we isolated a novel GmSHMT08 G357R carrying a missense mutation one residue away from the N358Y SNP found between Forrest and Essex, increasing the total of isolated Gmshmt08 EMS missense mutants to 17 ( Figure S4 ). It has recently been reported that the Forrest-specific polymorphic substitution N358Y impacted the mobility of a loop near the entrance of the (6S)-tetrahydrofolate-binding site, severely reducing its affinity for folate and dramatically impairing enzyme activity in Forrest GmSHMT08 [ 52 ]. Interestingly, an EMS mutation at the Gly357 residue resulted in the highest increase in female index (FI > 113%) ( Figure S4 ).…”

“…The E. coli eSHMT Gly124 (Forrest Gly132) is involved in a stacking interaction between the PLP pyridine ring and eSHMT His126 (ScSHMT His147, GmSHMT His134) [ 58 ]. The presence of this new haplotype may be the reason why Gmshmt08 G132D lost its catalytic activity, and could explain why the Forrest haplotype R130P is less catalytically active than the Essex Pro130 haplotype [ 8 , 52 ]. Considering proline has a conformational rigidity due to its direct incorporation of the α-carbon into its side chain, this may cause drastic conformational changes, interfering with this catalysis ( Figure 4 ).…”

“…It has been suggested that polymorphism residues that reside near to the ligand-binding sites may impair a key regulatory property of the GmSHMT08 enzyme [ 8 ]. In fact, the two Forrest polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the THF-binding site at the GmSHMT08 protein, resulting in reduced affinity for folate substrate, subsequently impairing the enzymatic activity of GmSHMT08 [ 52 ]. The isolated novel Gmshmt08 mutant from the EMS mutagenized Forrest soybean population G357R (FI = 113%) is located one residue away from the Forrest polymorphic substitution N358Y, and, therefore, is predicted to impact the THF site’s binding to folate.…”

“…Unlike mutations affecting the dimerization and tetramerization of the GmSHMT08 protein, the increase in the SCN female index in the S44F, G357R, and Y358N Gmshmt08 mutants is due to the loss of PLP catalysis and/or THF substrate binding [ 52 ]. These findings imply the role of both the GmSHMT08 structure and its enzymatic catalysis in SCN resistance.…”