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 α-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.

中文翻译：

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大豆丝氨酸羟甲基转移酶的 P285S 变体揭示了保守顺式脯氨酸的关键结构作用 8。


丝氨酸羟甲基转移酶 （SHMT） 在叶酸代谢中起关键作用，并且在所有生命界中都是保守的。SHMT 是一种吡哆醛 5'-磷酸 （PLP） 依赖性酶，可催化 L-丝氨酸和 （6S）-四氢叶酸转化为甘氨酸和 5,10-亚甲基四氢叶酸。SHMT 家族多个成员的晶体结构表明，该酶具有单个保守的顺式脯氨酸，位于活性位点附近。在这里，我们表征了影响大豆 SHMT8 中这种保守顺式脯氨酸的 Pro 到 Ser 氨基酸变体 （P285S）。P285S 被确定为影响大豆对农业病原体大豆孢囊线虫抗性的一组突变之一。我们发现，用丝氨酸取代 Pro285 消除了 PLP 介导的 SHMT8 催化活性，减少了叶酸结合，降低了酶的稳定性，并影响了酶在溶液中的二聚体-四聚体比率。1.9-2.2 Å 分辨率的晶体结构揭示了多肽链的局部重新排序，该序列延伸了一个 α 螺旋并将转折区域移动到活性位点。这导致 PLP 结合姿势受到严重扰动，其中辅因子的环翻转 ∼180°，同时保守的酶-PLP 相互作用丢失。多肽的附近区域变得无序，∼10 个残基的电子密度缺失就是证明。这些结构扰动与酶活性和叶酸结合的丧失一致，并强调了 Pro285 顺式肽在 SHMT 结构和功能中的重要作用。