Department of Biochemistry and Redox Biology Center, University of Nebraska-Lincoln, Lincoln, NE, 68588, USA.
Department of Pediatrics, Penn State College of Medicine, Hershey, PA, 17033, USA.
Arch Biochem Biophys. 2021 May 30;703:108852. doi: 10.1016/j.abb.2021.108852. Epub 2021 Mar 24.
Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P) releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.
吡咯啉-5-羧酸还原酶（人类中的 PYCR）通过使用 NAD(P)H 作为供氢体将 L-Δ-吡咯啉-5-羧酸（L-P5C）还原为 l-脯氨酸，催化 l-脯氨酸生物合成的最后一步。在人类中，已知有三种同工酶 PYCR1、PYCR2 和 PYCR3。最近的全基因组关联和临床研究表明，人类 PYCR2 的纯合突变导致出生后小头畸形和少突胶质细胞发育不良，包括少突胶质细胞发育不良 10 型。为了揭示人类 PYCR2 的生化和结构见解，我们对野生型酶以及先前在小头畸形和少突胶质细胞发育不良患者中发现的两种蛋白质变体 Arg119Cys 和 Arg251Cys 的稳态动力学进行了表征。使用 PYCR2 的动力学测量表明存在顺序结合机制，L-P5C 在 NAD(P)H 之前结合，NAD(P)在 L-Pro 之前释放。两种与疾病相关的变体均存在催化缺陷。取决于使用 NADPH 还是 NADH，R119C 蛋白变体的催化效率比野生型酶低 40 或 366 倍，而 R251C 蛋白变体的催化效率比野生型酶低 7 或 26 倍。此外，热稳定性和圆二色性测量表明 R251C 蛋白变体存在明显的折叠缺陷。这些结果与 Arg119Cys 和 Arg251Cys 参与疾病发病机制一致。
