Fe ion coordination direction and refresh mechanism in off-line αKG-dependent hydroxylase

In non-heme iron/α-ketoglutarate (αKG)-dependent enzyme which is one of the main forms of iron present in living organisms, αKG can bind in two different modes, “in-line” and “off-line”, depending on the orientation of its C-1 carboxyl. A classical mechanism involving a Fe(Ⅳ)=O intermediate has been proposed in in-line enzymes. However, no reasonable catalytic mechanism had been proposed for off-line αKG-dependent enzyme because αKG in this binding mode hinders the activated oxygen on Fe ion from approaching the substrate. In this study, we find the fixed coordination direction of Fe ion and propose a potential catalytic mechanism involving Fe ion release and refresh in non-heme αKG enzymes based on reaction intermediate crystal structures and enzyme assay of UbPH, an off-line αKG dependent trans-L-proline hydroxylase.

作为生物体内主要的铁存在形式之一的非血红素铁/α-酮戊二酸（αKG）依赖型酶，其αKG可依据其C-1羧基的取向，以直连式（in-line）与非直连式（off-line）两种不同模式结合。针对直连式结合模式的酶，学界已提出包含Fe(Ⅳ)=O中间体的经典催化机制。然而，针对非直连式结合模式的αKG依赖型酶，目前尚未提出合理的催化机制——因为该结合模式下的αKG会阻碍铁离子上的活化氧与底物接触。本研究基于非直连式αKG依赖型反式-L-脯氨酸羟化酶UbPH的反应中间体晶体结构与酶活实验数据，观测到铁离子存在固定的配位取向，并提出了一种涉及非血红素αKG酶中铁离子释放与更新的潜在催化机制。