L-Proline cis-4-hydroxylase (cis-P4H), a non-heme Fe²⁺/α-ketoglutarate-dependent dioxygenase (KDD), finds application in proline C4 hydroxylation and selective L-pipecolic acid (L-PA) C5 hydroxylation. Nonetheless, its regioselectivity is often ambiguous, yielding nearly equal amounts of cis-5/cis-3 hydroxylated L-PA isomers, posing challenges in separation and purification. In this study, we selected Kordia jejudonensis proline hydroxylase (KjPH) for modification and investigated the electrostatic effect's molecular mechanism on its regioselectivity. Through sequence and catalytic domain alignment of KjPH (cis-5/cis-3 = 20:1), SmP4H (cis-5/cis-3 = 1:1, from Sinorhizobium meliloti), and MlP4H (cis-5/cis-3 = 1:7, from Mesorhizobium loti), we identified four non-conserved key residues (Y35, S57, F95, and C97). We confirmed that F95 in KjPH plays a pivotal role in affecting regioselectivity. The single-site variant F95Y significantly enhanced regioselectivity, increasing the cis-5/cis-3 ratio from 20:1 to 55:1. Molecular dynamics simulations unveiled that the improved regioselectivity of the F95Y variant primarily resulted from the electrostatic repulsive interaction, which increased the distance between the substrate's C3 site and the Fe²⁺ catalytic core.

L-脯氨酸顺-4-羟化酶 ( cis -P4H) 是一种非血红素 Fe ²⁺ /α-酮戊二酸依赖性双加氧酶 (KDD)，可用于脯氨酸 C4 羟化和选择性 L-哌啶酸 (L-PA) C5羟基化。尽管如此，其区域选择性往往不明确，产生几乎等量的顺式-5/顺式-3 羟基化 L-PA 异构体，给分离和纯化带来了挑战。本研究选择Kordia jejudonensis脯氨酸羟化酶（KjPH）进行修饰，并研究静电效应对其区域选择性的分子机制。通过 KjPH ( cis -5/ cis -3 = 20:1)、SmP4H ( cis -5/ cis -3 = 1:1，来自苜蓿中华根瘤菌) 和 MlP4H ( cis -5/ cis - ) 的序列和催化结构域比对3 = 1:7，来自Mesorhizobium loti），我们鉴定了四个非保守关键残基（Y35、S57、F95 和 C97）。我们证实 KjPH 中的 F95 在影响区域选择性方面发挥着关键作用。单位点变体 F95Y 显着增强了区域选择性，将cis -5/ cis -3 比率从 20:1 增加到 55:1。^{分子动力学模拟表明，F95Y 变体的区域选择性提高主要是由于静电排斥相互作用，这增加了底物 C3 位点与 Fe 2+}催化核心之间的距离。