D-3-磷酸甘油酸脱氢酶(PHGDH)催化D-3-磷酸-甘油酸的NAD依赖性转化为3-磷酸羟基丙酮酸,L-丝氨酸(L-Ser)生物合成磷酸化途径的第一步。L-Ser在真核细胞中发挥不同的相关代谢作用:L-Ser代谢的改变与严重的神经系统疾病有关。人类PHGDH(hPHGDH),在溶液中显示同四聚体状态,由四个域组成,其中在C末端有两个调节域:天冬氨酸激酶-分支酸变位酶-tyrA预苯酸脱氢酶(ACT)和变构底物结合(ASB)域。hPHGDH的结构仅对于截短的,二聚体形式具有包含底物和辅因子结合域的N末端。使用AlphaFold结合分子动力学精修生成四聚体hPHGDH的模型集合。通过分析四聚体界面的亚基间相互作用,选择残基F418,L478,P479,R454和Y495,并通过丙氨酸扫描诱变方法研究它们的作用。F418A变体修饰了推定的ASB,稍微改变了活动,四聚体状态的蛋白质部分,和蛋白质稳定性;它似乎与二聚体识别相关,以产生四聚体寡聚体。相反,R454A,L478A,P479A,和Y495A变体(ACT结构域)确定四聚体组装的丢失,导致低稳定性和错误折叠,触发聚合并妨碍活动。预测的四聚体界面似乎是由ACT结构域的残基介导的,四聚体的形成似乎对hPHGDH的正确折叠至关重要,which,反过来,对于稳定性和功能性都至关重要。
D-3-phosphoglycerate dehydrogenase (PHGDH) catalyzes the NAD+-dependent conversion of D-3-phospho-glycerate to 3-phosphohydroxypyruvate, the first step in the phosphorylated pathway for L-serine (L-Ser) biosynthesis. L-Ser plays different relevant metabolic roles in eukaryotic cells: alterations in L-Ser metabolism have been linked to serious neurological disorders. The human PHGDH (hPHGDH), showing a homotetrameric state in solution, is made of four domains, among which there are two regulatory domains at the C-terminus: the aspartate kinase-chorismate mutase-tyrA prephenate dehydrogenase (ACT) and allosteric substrate-binding (ASB) domains. The structure of hPHGDH was solved only for a truncated, dimeric form harboring the N-terminal end containing the substrate and the cofactor binding domains. A model ensemble of the tetrameric hPHGDH was generated using AlphaFold coupled with molecular dynamics refinement. By analyzing the inter-subunit interactions at the tetrameric interface, the residues F418, L478, P479, R454, and Y495 were selected and their role was studied by the alanine-scanning mutagenesis approach. The F418A variant modifies the putative ASB, slightly alters the activity, the fraction of protein in the tetrameric state, and the protein stability; it seems relevant in dimers\' recognition to yield the tetrameric oligomer. On the contrary, the R454A, L478A, P479A, and Y495A variants (ACT domain) determine a loss of the tetrameric assembly, resulting in low stability and misfolding, triggering the aggregation and hampering the activity. The predicted tetrameric interface seems mediated by residues at the ACT domain, and the tetramer formation seems crucial for proper folding of hPHGDH, which, in turn, is essential for both stability and functionality.