Department of Biochemical Sciences "A. Rossi Fanelli", Sapienza University of Rome, Rome, Italy.
Istituto Pasteur of Italy - Fondazione Cenci Bolognetti, Rome, Italy.
Sci Rep. 2017 Sep 15;7(1):11704. doi: 10.1038/s41598-017-11948-w.
The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5'-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.
乙醛酸氨基转移酶（AGT）是一种肝细胞特异性吡哆醛-5'-磷酸（PLP）依赖性酶，可将 L-丙氨酸和乙醛酸转氨基为甘氨酸和丙酮酸，从而解毒乙醛酸并防止组织中病理性草酸沉淀。在广泛接受的氨基转移酶家族催化机制中，与 PLP 结合的赖氨酸作为逐步 1,3-质子转移的催化剂，将外部亚胺醛转化为亚胺酮。这一步需要由 PLP 吡啶氮的保守天冬氨酸质子化，以增强其稳定碳负离子中间体的能力。天冬氨酸残基还负责内部亚胺醛的显著几何变形，这对于催化至关重要。我们展示了人 AGT 的结构，其中完整的 X 射线光还原 Schiff 碱已经发生。这一结果，以及两个保守天冬氨酸致病性变体（D183N）的晶体结构和转亚胺化步骤的分子建模，使我们提出相反力的相互作用，我们称之为弹簧机制，在催化过程中精细地调整 PLP 几何形状，对于将外部亚胺醛移动到正确的位置以进行 1,3-质子转移至关重要。
