Daudu Oseeyi I, Meeks Kaylen R, Zhang Lu, Seravalli Javier, Tanner John J, Becker Donald F
Department of Biochemistry, Redox Biology Center, University of Nebraska, Lincoln, Nebraska 68588, United States.
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, United States.
ACS Omega. 2023 Jan 10;8(3):3509-3519. doi: 10.1021/acsomega.2c07788. eCollection 2023 Jan 24.
Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ-pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity.
吡咯啉-5-羧酸还原酶（PYCR）是一种脯氨酸生物合成酶，催化依赖于NAD(P)H将Δ-吡咯啉-5-羧酸（P5C）还原为脯氨酸。人类有三种PYCR同工型，其中PYCR1在不同类型的癌症中常上调。在此，我们研究了PYCR1的Thr171Met变体的生化和结构特性，该变体在恶性黑色素瘤和肺腺癌患者中发现。尽管PYCR1与癌症进展密切相关，但尚未有关于癌症患者中PYCR1变体特征的报道。Thr171在所有三种PYCR同工酶中保守，且位于P5C底物结合位点附近。我们发现氨基酸替换不影响热稳定性，但对PYCR1的催化活性有深远影响。当P5C为可变底物时，PYCR1变体T171M的 比野生型PYCR1低100至200倍，当NAD(P)H变化时低10至25倍。T171M的1.84 Å分辨率X射线晶体结构显示，甲硫氨酸侧链侵入P5C底物结合位点，表明催化缺陷是由于空间冲突阻止P5C达到从NAD(P)H进行氢化物转移的最佳构象。这些结果表明，在癌症中与T171M相关的对PYCR1功能的任何影响并非源于催化活性增加。
