Expression and kinetic characterization of PYCR3.
Expression and kinetic characterization of PYCR3.
复制标题
PYCR3 的表达和动力学特征。
DOI：
10.1016/j.abb.2022.109468
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发表时间：
2023
影响因子：
3.9
通讯作者：
Tanner,JohnJ
中科院分区：
文献类型：
--
作者：
Meeks,KaylenR;Tanner,JohnJ
PYCR是脯氨酸生物合成酶，其催化人中Δ1-吡咯啉-5-羧酸（P5 C）的NAD（P）H依赖性还原为脯氨酸。PYCRs -特别是PYCR 1-在许多类型的癌症中上调，并且与癌细胞的代谢改变有关。在PYCR的三种同种型中，PYCR 3仍然是研究最少的，部分原因是缺乏稳健的重组表达。在此，我们描述了可溶性SUMO-PYCR 3在大肠杆菌中的表达，融合蛋白的纯化和SUMO标签的去除的过程。PYCR 3在NADPH或NADH作为辅酶的情况下具有活性。通过改变L-P5 C和NADH的浓度获得的双底物动力学测量，沿着l-脯氨酸的产物抑制数据，表明随机有序的双双机制。筛选一组19种脯氨酸类似物的抑制作用，并测量五种筛选的化合物的竞争性抑制动力学（用L-P5 C），包括N-甲酰基-1-脯氨酸，一种经验证的PYCR 1抑制剂。发现N-甲酰基-1-脯氨酸对PYCR 1的选择性是PYCR 3的十倍。SUMO-PYCR 3表达系统可用于检测PYCR 1抑制剂的亚型特异性。
PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 inEscherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data forl-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors.